Calcitonin gene-related peptide (cgrp) antagonist antibodies

ABSTRACT

This application provides isolated antibodies, and antigen-binding fragments thereof, that specifically bind Calcitonin Gene Related Peptide (CGRP). These anti-CGRP antibodies, or antigen-binding fragments thereof, have a high affinity for CGRP, function to inhibit CGRP, are less immunogenic compared to their unmodified parent antibodies in a given species (e.g., a human) and can be used to treat CGRP-associated disorders, while avoiding the adverse side effects associated with the current CGRP antagonist therapies.

RELATED PATENT APPLICATIONS

This application claims benefit of U.S. Provisional Application No.62/634,643, filed on Feb. 23, 2018, incorporated in its entirety byreference herein.

TECHNICAL FIELD

Calcitonin Gene-Related Peptide (CGRP) is a multifunctional neuropeptideof 37 amino acids in length secreted by the nerves of the central andperipheral nervous systems. Two forms of CGRP, the CGRP-α and CGRP-βforms, exist in humans and have similar activities. CGRP-α and CGRP-βdiffer by three amino acids in humans, and are derived from differentgenes. The CGRP family of peptides includes amylin, adrenomedullin, andcalcitonin, although each has distinct receptors and biologicalactivities (Doods, H., Curr. Op. Invest. Drugs, 2(9):1261-68 (2001)).

CGRP is a potent peptide vasodilator postulated to be involved inseveral of the pathophysiological processes, including dilation ofcerebral and dural blood vessels, release of inflammatory mediators frommast cells, and transmission of nociceptive information fromintracranial blood vessels to the nervous system, and can function inthe transmission of pain (Durham P L, N Engl J Med., 350:1073-1074,2004)). Several findings in support a role of CGRP in migraine. First,serum concentrations of CGRP are elevated during migraine attacks aswell as episodes of other neurovascular headache types such as clusterheadache. Furthermore, relief of migraine pain by triptans coincideswith reduction in or normalization of CGRP concentrations in blood. Thefindings that selective CGRP receptor antagonists reduce vasodilationand neurogenic inflammation and confer clinical benefit in migrainefurther support an integral role of CGRP in migraine (Durham P L,Headache, June 46(1): S3-S8, 2006)).

Increased levels of CGRP have also been reported in temporomandibularjoint disorder patients as well as a variety of other diseases such ascardiac failure, hypertension, and sepsis (Goto et al., Ann NY Acad Sci.657: 194-203, 1992; Joyce et al., Surgery. 108 (6): 1097-101, 1990)).There is mounting evidence to suggest that CGRP is beneficial inpreventing the development of hypertension and cardiovascularpathologies associated with hypertension, diabetes, obesity andarthritis (Russell et al., Physiological Reviews. 94(4): 1099-1142,2014)). Fremanezumab, a humanized monoclonal antibody directed againstCGRP alpha and beta, is in phase III clinical trials for clusterheadache/migraine (see, e.g, ClinicalTrials NCT02964338).

Due to the perceived involvement of CGRP in these diseases anddisorders, there remains a need in the art for compositions and methodsuseful for preventing or treating diseases and disorders associated withCGRP, while avoiding adverse side effects. Specifically, there remains aneed in the art for compositions or methods useful for preventing ortreating CGRP-associated disorders such as headaches, hot flushes,chronic pain, type II diabetes mellitus, functional bowel disorders oran inflammatory bowel diseases, diarrhea, psoriasis, pain and itchassociated with arthritis and skin disease, cardiovascular disorders,and hemodynamic derangement associated with endotoxemia, sepsis,obesity, diabetes and arthritis, while avoiding adverse side effects.

DISCLOSURE OF THE INVENTION

In accordance with the present invention, there are provided isolatedantibodies, and antigen-binding fragments thereof, that specificallybind calcitonin gene-related peptide (CGRP). These CGRP antibodies, orantigen-binding fragments thereof, have a high affinity for CGRP,function to inhibit CGRP, are less immunogenic compared to theirunmodified parent antibodies in a given species (e.g., in a human), andcan be used to treat CGRP-associated disorders.

In various embodiments, the antibody or antigen-binding fragment isselected from a human antibody, a humanized antibody, chimeric antibody,a monoclonal antibody, a polyclonal antibody, a recombinant antibody, asingle chain antibody, a diabody, a triabody, a tetrabody, a Fabfragment, a Fab′ fragment, a Fab₂ fragment, a F(ab)′₂ fragment, a domainantibody, an IgD antibody, an IgE antibody, an IgM antibody, an IgG1antibody, an IgG2 antibody, an IgG3 antibody, an IgG4 antibody, or anIgG4 antibody having at least one mutation in the hinge region thatalleviates a tendency to form intra H-chain disulfide bonds. In variousembodiments, the antibody is a chimeric antibody. In variousembodiments, the antibody is a humanized antibody. In variousembodiments, the antibody is a fully human antibody. In variousembodiments, isolated antibodies, and antigen-binding fragments thereof,that have a high affinity for the human CGRP of SEQ ID NO: 1 areprovided.

In various embodiments, the antibody or antigen-binding fragment bindsto CGRP protein with a dissociation constant (K_(D)) of at least about1×10⁻⁶ M, at least about 1×10⁻⁷ M, at least about 1×10⁻⁸ M, at leastabout 1×10⁻⁹ M, at least about 1×10⁻¹⁰ M, at least about 1×10⁻¹¹ M, orat least about 1×10⁻¹² M.

In various embodiments, an isolated antibody or antigen-binding fragmentthereof of the present invention binds to human CGRP and compriseseither: (a) a light chain CDR3 sequence identical, substantiallyidentical or substantially similar to a CDR3 sequence selected from SEQID NOs: 14-16; (b) a heavy chain CDR3 sequence identical, substantiallyidentical or substantially similar to a CDR3 sequence selected from SEQID NOs: 6-8; or (c) the light chain CDR3 sequence of (a) and the heavychain CDR3 sequence of (b).

In various embodiments, the isolated antibody or antigen-bindingfragment further comprises an amino acid sequence selected from: (d) alight chain CDR1 sequence identical, substantially identical orsubstantially similar to a CDR1 sequence selected from SEQ ID NOs: 9-11;(e) a light chain CDR2 sequence identical, substantially identical orsubstantially similar to a CDR2 sequence selected from SEQ ID NOs:12-13; (f) a heavy chain CDR1 sequence identical, substantiallyidentical or substantially similar to a CDR1 sequence selected from SEQID NO: 2; (g) a heavy chain CDR2 sequence identical, substantiallyidentical or substantially similar to a CDR2 sequence selected from SEQID NOs: 3-5; (h) the light chain CDR1 sequence of (d) and the heavychain CDR1 sequence of (f); or (i) the light chain CDR2 sequence of (e)and the heavy chain CDR2 sequence of (g).

In various embodiments, the isolated human monoclonal antibody orantigen-binding fragment thereof of the present invention binds to humanCGRP and comprises: (a) a light chain CDR1 sequence identical,substantially identical or substantially similar to a CDR1 sequenceselected from SEQ ID NOs: 9-11; (b) a light chain CDR2 sequenceidentical, substantially identical or substantially similar to a CDR2sequence selected from SEQ ID NOs: 12-13; (c) a light chain CDR3sequence identical, substantially identical or substantially similar toa CDR3 sequence selected from SEQ ID NOs: 14-16; (d) a heavy chain CDR1sequence identical, substantially identical or substantially similar toa CDR1 sequence selected from SEQ ID NO: 2; (e) a heavy chain CDR2sequence identical, substantially identical or substantially similar toa CDR2 sequence selected from SEQ ID NOs: 3-5; and (f) a heavy chainCDR3 sequence identical, substantially identical or substantiallysimilar to a CDR3 sequence selected from SEQ ID NOs: 6-8.

In various embodiments, the isolated human monoclonal antibody orantigen-binding fragment thereof of the present invention binds to humanCGRP and comprises: (a) a light chain CDR1 sequence identical,substantially identical or substantially similar to SEQ ID NO: 11; (b) alight chain CDR2 sequence identical, substantially identical orsubstantially similar to SEQ ID NO: 14; (c) a light chain CDR3 sequenceidentical, substantially identical or substantially similar to SEQ IDNO: 17; (d) a heavy chain CDR1 sequence identical, substantiallyidentical or substantially similar to SEQ ID NO: 2; (e) a heavy chainCDR2 sequence identical, substantially identical or substantiallysimilar to SEQ ID NO: 5; and (f) a heavy chain CDR3 sequence identical,substantially identical or substantially similar to SEQ ID NO: 8.

In various embodiments, the isolated human monoclonal antibody orantigen-binding fragment thereof of the present invention binds to humanCGRP and comprises: (a) a light chain CDR1 sequence identical,substantially identical or substantially similar to SEQ ID NO: 12; (b) alight chain CDR2 sequence identical, substantially identical orsubstantially similar to SEQ ID NO: 15; (c) a light chain CDR3 sequenceidentical, substantially identical or substantially similar to SEQ IDNO: 18; (d) a heavy chain CDR1 sequence identical, substantiallyidentical or substantially similar to SEQ ID NO: 3; (e) a heavy chainCDR2 sequence identical, substantially identical or substantiallysimilar to SEQ ID NO: 6; and (f) a heavy chain CDR3 sequence identical,substantially identical or substantially similar to SEQ ID NO: 9.

In various embodiments, the isolated human monoclonal antibody orantigen-binding fragment thereof of the present invention binds to humanCGRP and comprises: (a) a light chain CDR1 sequence identical,substantially identical or substantially similar to SEQ ID NO: 13; (b) alight chain CDR2 sequence identical, substantially identical orsubstantially similar to SEQ ID NO: 16; (c) a light chain CDR3 sequenceidentical, substantially identical or substantially similar to SEQ IDNO: 19; (d) a heavy chain CDR1 sequence identical, substantiallyidentical or substantially similar to SEQ ID NO: 4; (e) a heavy chainCDR2 sequence identical, substantially identical or substantiallysimilar to SEQ ID NO: 7; and (f) a heavy chain CDR3 sequence identical,substantially identical or substantially similar to SEQ ID NO: 10.

In various embodiments, an isolated antibody or antigen-binding fragmentthereof of the present invention binds to human CGRP and compriseseither: (a) a heavy and/or light chain variable domain(s), the variabledomain(s) having a set of three light chain CDR1, CDR2, and CDR3identical, substantially identical or substantially similar to SEQ IDNOs: 9-11, 12-13, and 14-16, and/or a set of three heavy chain CDR1,CDR2, and CDR3 identical, substantially identical or substantiallysimilar to SEQ ID NO: 2, 3-5, and 6-8; and (b) a set of four variableregion framework regions from a human immunoglobulin (IgG). In variousembodiments, the antibody can optionally include a hinge region. Invarious embodiments, the framework regions are chosen from humangermline exon X_(H), J_(H), V_(K) and J_(K) sequences. In variousembodiments, the antibody is a fully humanized antibody. In variousembodiments, the antibody is a fully human antibody.

In various embodiments, an isolated antibody or antigen-binding fragmentthereof of the present invention binds to human CGRP and comprises theheavy chain variable region having the amino acid sequence set forth inSEQ ID NO: 17 and the light chain variable region having the amino acidsequence set forth in SEQ ID NO: 23. In various embodiments, an isolatedantibody or antigen-binding fragment thereof of the present inventionbinds to human CGRP and comprises the heavy chain variable region havingthe amino acid sequence set forth in SEQ ID NO: 19 and the light chainvariable region having the amino acid sequence set forth in SEQ ID NO:25. In various embodiments, an isolated antibody or antigen-bindingfragment thereof of the present invention binds to human CGRP andcomprises the heavy chain variable region having the amino acid sequenceset forth in SEQ ID NO: 21 and the light chain variable region havingthe amino acid sequence set forth in SEQ ID NO: 27.

In various embodiments, the isolated antibody or antigen-bindingfragment, when bound to human CGRP: (a) binds to human CGRP withsubstantially the same or greater Kd as a reference antibody; (b)competes for binding to human CGRP with said reference antibody; or (c)is less immunogenic in a human subject than said reference antibody,wherein said reference antibody comprises a combination of heavy chainvariable domain and light chain variable domain sequences selected fromSEQ ID NOs: 17/23, 19/25 and 21/27.

In various embodiments, an isolated chimeric antibody or antigen-bindingfragment thereof of the present invention binds to human CGRP andcomprises a heavy chain having a sequence identical, substantiallyidentical or substantially similar to SEQ ID NO: 29, and a light chainhaving the sequence identical, substantially identical or substantiallysimilar to SEQ ID NO: 31.

In various embodiments, an isolated humanized antibody orantigen-binding fragment thereof of the present invention binds to humanCGRP and comprises a heavy chain variable region having a sequenceidentical, substantially identical or substantially similar to SEQ IDNOs: 33, 35, 37 and 39, and a light chain variable region having thesequence identical, substantially identical or substantially similar toSEQ ID NOs: 34, 36, 38 and 40. In various embodiments the antibody is ahumanized antibody or antigen-binding fragment thereof which comprisesthe heavy chain variable region having the amino acid sequence set forthin SEQ ID NO: 33 and the light chain variable region having the aminoacid sequence set forth in SEQ ID NO: 34. In various embodiments theantibody is a humanized antibody or antigen-binding fragment thereofwhich comprises the heavy chain variable region having the amino acidsequence set forth in SEQ ID NO: 33 and the light chain variable regionhaving the amino acid sequence set forth in SEQ ID NO: 36. In variousembodiments the antibody is a humanized antibody or antigen-bindingfragment thereof which comprises the heavy chain variable region havingthe amino acid sequence set forth in SEQ ID NO: 33 and the light chainvariable region having the amino acid sequence set forth in SEQ ID NO:38. In various embodiments the antibody is a humanized antibody orantigen-binding fragment thereof which comprises the heavy chainvariable region having the amino acid sequence set forth in SEQ ID NO:33 and the light chain variable region having the amino acid sequenceset forth in SEQ ID NO: 40. In various embodiments the antibody is ahumanized antibody or antigen-binding fragment thereof which comprisesthe heavy chain variable region having the amino acid sequence set forthin SEQ ID NO: 35 and the light chain variable region having the aminoacid sequence set forth in SEQ ID NO: 34. In various embodiments theantibody is a humanized antibody or antigen-binding fragment thereofwhich comprises the heavy chain variable region having the amino acidsequence set forth in SEQ ID NO: 35 and the light chain variable regionhaving the amino acid sequence set forth in SEQ ID NO: 36. In variousembodiments the antibody is a humanized antibody or antigen-bindingfragment thereof which comprises the heavy chain variable region havingthe amino acid sequence set forth in SEQ ID NO: 35 and the light chainvariable region having the amino acid sequence set forth in SEQ ID NO:38. In various embodiments the antibody is a humanized antibody orantigen-binding fragment thereof which comprises the heavy chainvariable region having the amino acid sequence set forth in SEQ ID NO:35 and the light chain variable region having the amino acid sequenceset forth in SEQ ID NO: 40. In various embodiments the antibody is ahumanized antibody or antigen-binding fragment thereof which comprisesthe heavy chain variable region having the amino acid sequence set forthin SEQ ID NO: 37 and the light chain variable region having the aminoacid sequence set forth in SEQ ID NO: 34. In various embodiments theantibody is a humanized antibody or antigen-binding fragment thereofwhich comprises the heavy chain variable region having the amino acidsequence set forth in SEQ ID NO: 37 and the light chain variable regionhaving the amino acid sequence set forth in SEQ ID NO: 36. In variousembodiments the antibody is a humanized antibody or antigen-bindingfragment thereof which comprises the heavy chain variable region havingthe amino acid sequence set forth in SEQ ID NO: 37 and the light chainvariable region having the amino acid sequence set forth in SEQ ID NO:38. In various embodiments the antibody is a humanized antibody orantigen-binding fragment thereof which comprises the heavy chainvariable region having the amino acid sequence set forth in SEQ ID NO:37 and the light chain variable region having the amino acid sequenceset forth in SEQ ID NO: 40. In various embodiments the antibody is ahumanized antibody or antigen-binding fragment thereof which comprisesthe heavy chain variable region having the amino acid sequence set forthin SEQ ID NO: 39 and the light chain variable region having the aminoacid sequence set forth in SEQ ID NO: 34. In various embodiments theantibody is a humanized antibody or antigen-binding fragment thereofwhich comprises the heavy chain variable region having the amino acidsequence set forth in SEQ ID NO: 39 and the light chain variable regionhaving the amino acid sequence set forth in SEQ ID NO: 36. In variousembodiments the antibody is a humanized antibody or antigen-bindingfragment thereof which comprises the heavy chain variable region havingthe amino acid sequence set forth in SEQ ID NO: 39 and the light chainvariable region having the amino acid sequence set forth in SEQ ID NO:38. In various embodiments the antibody is a humanized antibody orantigen-binding fragment thereof which comprises the heavy chainvariable region having the amino acid sequence set forth in SEQ ID NO:39 and the light chain variable region having the amino acid sequenceset forth in SEQ ID NO: 40.

In various embodiments, an isolated humanized antibody orantigen-binding fragment thereof of the present invention binds to humanCGRP and comprises a heavy chain having a sequence identical,substantially identical or substantially similar to SEQ ID NOs: 43, 45,47 and 49, and a light chain variable region having the sequenceidentical, substantially identical or substantially similar to SEQ IDNOs: 49, 51, 53 and 55. In various embodiments, an isolated humanizedantibody or antigen-binding fragment thereof of the present inventionbinds to human CGRP and comprises the heavy chain sequence set forth inSEQ ID NO: 41, and the light chain sequence set forth in SEQ ID NO: 49.In various embodiments, an isolated humanized antibody orantigen-binding fragment thereof of the present invention binds to humanCGRP and comprises the heavy chain sequence set forth in SEQ ID NO: 41,and the light chain sequence set forth in SEQ ID NO: 51. In variousembodiments, an isolated humanized antibody or antigen-binding fragmentthereof of the present invention binds to human CGRP and comprises theheavy chain sequence set forth in SEQ ID NO: 41, and the light chainsequence set forth in SEQ ID NO: 53. In various embodiments, an isolatedhumanized antibody or antigen-binding fragment thereof of the presentinvention binds to human CGRP and comprises the heavy chain sequence setforth in SEQ ID NO: 41, and the light chain sequence set forth in SEQ IDNO: 55. In various embodiments, an isolated humanized antibody orantigen-binding fragment thereof of the present invention binds to humanCGRP and comprises the heavy chain sequence set forth in SEQ ID NO: 43,and the light chain sequence set forth in SEQ ID NO: 49. In variousembodiments, an isolated humanized antibody or antigen-binding fragmentthereof of the present invention binds to human CGRP and comprises theheavy chain sequence set forth in SEQ ID NO: 43, and the light chainsequence set forth in SEQ ID NO: 51. In various embodiments, an isolatedhumanized antibody or antigen-binding fragment thereof of the presentinvention binds to human CGRP and comprises the heavy chain sequence setforth in SEQ ID NO: 43, and the light chain sequence set forth in SEQ IDNO: 53. In various embodiments, an isolated humanized antibody orantigen-binding fragment thereof of the present invention binds to humanCGRP and comprises the heavy chain sequence set forth in SEQ ID NO: 43,and the light chain sequence set forth in SEQ ID NO: 55. In variousembodiments, an isolated humanized antibody or antigen-binding fragmentthereof of the present invention binds to human CGRP and comprises theheavy chain sequence set forth in SEQ ID NO: 45, and the light chainsequence set forth in SEQ ID NO: 49. In various embodiments, an isolatedhumanized antibody or antigen-binding fragment thereof of the presentinvention binds to human CGRP and comprises the heavy chain sequence setforth in SEQ ID NO: 45, and the light chain sequence set forth in SEQ IDNO: 51. In various embodiments, an isolated humanized antibody orantigen-binding fragment thereof of the present invention binds to humanCGRP and comprises the heavy chain sequence set forth in SEQ ID NO: 45,and the light chain sequence set forth in SEQ ID NO: 53. In variousembodiments, an isolated humanized antibody or antigen-binding fragmentthereof of the present invention binds to human CGRP and comprises theheavy chain sequence set forth in SEQ ID NO: 45, and the light chainsequence set forth in SEQ ID NO: 55. In various embodiments, an isolatedhumanized antibody or antigen-binding fragment thereof of the presentinvention binds to human CGRP and comprises the heavy chain sequence setforth in SEQ ID NO: 47, and the light chain sequence set forth in SEQ IDNO: 49. In various embodiments, an isolated humanized antibody orantigen-binding fragment thereof of the present invention binds to humanCGRP and comprises the heavy chain sequence set forth in SEQ ID NO: 47,and the light chain sequence set forth in SEQ ID NO: 51. In variousembodiments, an isolated humanized antibody or antigen-binding fragmentthereof of the present invention binds to human CGRP and comprises theheavy chain sequence set forth in SEQ ID NO: 47, and the light chainsequence set forth in SEQ ID NO: 53. In various embodiments, an isolatedhumanized antibody or antigen-binding fragment thereof of the presentinvention binds to human CGRP and comprises the heavy chain sequence setforth in SEQ ID NO: 47, and the light chain sequence set forth in SEQ IDNO: 55.

In another aspect, the present invention relates to a pharmaceuticalcomposition comprising an isolated antibody or antigen-binding fragmentof the present invention in admixture with a pharmaceutically acceptablecarrier. In various embodiments, the pharmaceutical compositioncomprises an isolated human antibody in admixture with apharmaceutically acceptable carrier. In various embodiments, thepharmaceutical composition is formulated for administration via a routeselected from the group consisting of subcutaneous injection,intraperitoneal injection, intramuscular injection, intrasternalinjection, intravenous injection, intraarterial injection, intrathecalinjection, intraventricular injection, intraurethral injection,intracranial injection, intrasynovial injection or via infusions.

In another aspect, the present invention relates to methods of treatinga subject suffering from a CGRP-associated disorder, comprisingadministering to said subject a therapeutically effective amount of anantibody or antigen-binding fragment thereof of the present invention.In various embodiments, the subject is a human subject. In variousembodiments, the CGRP-associated disorder is selected from the groupconsisting of headaches, hot flushes, chronic pain, type II diabetesmellitus, functional bowel disorders or an inflammatory bowel diseases,diarrhea, psoriasis, pain and itch associated with arthritis and skindisease, cardiovascular disorders, and hemodynamic derangementassociated with endotoxemia, sepsis, obesity, diabetes and arthritis.

In various embodiments, the CGRP-associated disorder is a headacheselected from the group consisting of migraine with or without aura;hemiplegic migraine; cluster headaches; migrainous neuralgia; chronicheadaches; tension headaches; headaches resulting from other medicalconditions (such as infection or increased pressure in the skull due toa tumor); chronic paroxysmal hemicrania; miscellaneous headacheunassociated with a structural lesion; headache associated with anon-vascular intracranial disorder; headache associated with theadministration of a substance or its withdrawal; headache associatedwith noncephalic infection; headache associated with a metabolicdisorder; headache associated with a disorder of the cranium, neck,eyes, ears, nose, sinuses, teeth, mouth or other facial or cranialstructure; cranial neuralgias; and nerve trunk pain and deafferentiationpain.

In another aspect, the present invention relates to combinationtherapies designed to treat a CGRP-associated disorder, comprisingadministering to the subject a therapeutically effective amount of anisolated antibody or antigen-binding fragment of the present invention,and b) one or more additional agent useful for treating the specificCGRP-associated disorder. In various embodiments, the CGRP-associateddisorder is selected from the group consisting of headaches, hotflushes, chronic pain, type II diabetes mellitus, functional boweldisorders or an inflammatory bowel diseases, diarrhea, psoriasis, painand itch associated with arthritis and skin disease, cardiovasculardisorders, and hemodynamic derangement associated with endotoxemia,sepsis, obesity, diabetes and arthritis.

In various embodiments, the CGRP-associated disorder is a headache andthe one or more additional agent is selected from the group consistingof: 5-HT1-like agonists (and agonists acting at other 5-HT1 sites), andnon-steroidal anti-inflammatory drugs (NSAIDs). In various embodiments,the CGRP-associated disorder is hot flashes and the one or moreadditional agent is a hormone-based treatment.

In another aspect, the antibodies or antigen-binding fragments disclosedherein may be covalently linked to (or otherwise stably associated with)an additional functional moiety, such as a label or a moiety thatconfers desirable pharmacokinetic properties. In various embodiments,the label is selected from the group consisting of: a fluorescent label,a radioactive label, and a label having a distinctive nuclear magneticresonance signature.

In another aspect, the present invention provides a method for detectingin vitro or in vivo the presence of human CGRP peptide in a sample,e.g., for diagnosing a human CGRP-associated disorder.

In another aspect, isolated nucleic acids comprising the polynucleotidesequence that encodes the antibodies or antigen-binding fragmentsdisclosed herein are provided. Also provided are expression vectorscomprising the nucleic acid of the present invention. Also provided areisolated cells comprising the expression vectors of the invention. Invarious embodiments, the cell is a host cell comprising an expressionvector of the present invention. In various embodiments, the cell is ahybridoma, wherein the chromosome of the cell comprises a nucleic acidof the present invention. Further provided is a method of making theantibody or antigen-binding fragment of the present invention comprisingculturing or incubating the cell under conditions that allow the cell toexpress the antibody or antigen-binding fragment of the presentinvention.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 depicts line graphs depicting the results of the evaluation ofthe 15 murine monoclonal antibodies in a human CGRP binding avidityassay (ELISA) and a murine CGRP crossreactivity assay (ELISA).

FIG. 2 depicts line graphs depicting the results of the evaluation ofthe 15 murine monoclonal antibodies in a SK-N-MC cells CGRP assay.

FIG. 3 depicts line graphs depicting the results of the evaluation ofthe 4 humanized monoclonal antibodies as compared to the parental murinemonoclonal antibody and a non-CGRP binding human monoclonal antibody(REMD477) in a SK-N-MC cells CGRP assay.

MODE(S) OF CARRYING OUT THE INVENTION

The present invention relates to antigen binding proteins such asantibodies, or antigen-binding fragments thereof that specifically bindto human CGRP. In one aspect, there are provided isolated antibodies,and antigen-binding fragments thereof, that specifically bind CGRP, havea high affinity for CGRP, function to inhibit CGRP, are less immunogeniccompared to their unmodified parent antibodies in a given species (e.g.,a human), and can be used to treat human disorders mediated by CGRP.Also provided are nucleic acid molecules, and derivatives and fragmentsthereof, comprising a sequence of polynucleotides that encode all or aportion of a polypeptide that binds to CGRP, such as a nucleic acidencoding all or part of an anti-CGRP antibody, antibody fragment, orantibody derivative. Also provided are vectors and plasmids comprisingsuch nucleic acids, and cells or cell lines comprising such nucleicacids and/or vectors and plasmids. Also provided are methods of making,identifying, or isolating antigen binding proteins that bind to humanCGRP, such as anti-CGRP antibodies, methods of determining whether anantigen binding protein binds to CGRP, methods of making compositions,such as pharmaceutical compositions, comprising an antigen bindingprotein that binds to human CGRP, and methods for administering anantibody, or antigen-binding fragment thereof that binds CGRP to asubject, for example, methods for treating a condition mediated by CGRP.

Definitions

Unless otherwise defined herein, scientific and technical terms used inconnection with the present invention shall have the meanings that arecommonly understood by those of ordinary skill in the art. Further,unless otherwise required by context, singular terms shall includepluralities and plural terms shall include the singular. Generally,nomenclatures used in connection with, and techniques of, cell andtissue culture, molecular biology, immunology, microbiology, geneticsand protein and nucleic acid chemistry and hybridization describedherein are those commonly used and well known in the art. The methodsand techniques of the present invention are generally performedaccording to conventional methods well known in the art and as describedin various general and more specific references that are cited anddiscussed throughout the present specification unless otherwiseindicated. See, e.g., Green and Sambrook, Molecular Cloning: ALaboratory Manual, 4th ed., Cold Spring Harbor Laboratory Press, ColdSpring Harbor, N.Y. (2012), incorporated herein by reference. Enzymaticreactions and purification techniques are performed according tomanufacturer's specifications, as commonly accomplished in the art or asdescribed herein. The nomenclature used in connection with, and thelaboratory procedures and techniques of, analytical chemistry, syntheticorganic chemistry, and medicinal and pharmaceutical chemistry describedherein are those commonly used and well known in the art. Standardtechniques are used for chemical syntheses, chemical analyses,pharmaceutical preparation, formulation, and delivery, and treatment ofsubjects.

Polynucleotide and polypeptide sequences are indicated using standardone- or three-letter abbreviations. Unless otherwise indicated,polypeptide sequences have their amino termini at the left and theircarboxy termini at the right, and single-stranded nucleic acidsequences, and the top strand of double-stranded nucleic acid sequences,have their 5′ termini at the left and their 3′ termini at the right. Aparticular section of a polypeptide can be designated by amino acidresidue number such as amino acids 80 to 119, or by the actual residueat that site such as Ser80 to Ser119. A particular polypeptide orpolynucleotide sequence also can be described based upon how it differsfrom a reference sequence. Polynucleotide and polypeptide sequences ofparticular light and heavy chain variable domains are designated L1(“light chain variable domain 1”) and H1 (“heavy chain variable domain1”). Antibodies comprising a light chain and heavy chain are indicatedby combining the name of the light chain and the name of the heavy chainvariable domains. For example, “L4H7,” indicates, for example, anantibody comprising the light chain variable domain of L4 and the heavychain variable domain of H7.

The term “antibody” is used herein to refer to a protein comprising oneor more polypeptides substantially or partially encoded byimmunoglobulin genes or fragments of immunoglobulin genes and havingspecificity to a tumor antigen or specificity to a moleculeoverexpressed in a pathological state. The recognized immunoglobulingenes include the kappa, lambda, alpha, gamma, delta, epsilon and muconstant region genes, as well as subtypes of these genes and myriad ofimmunoglobulin variable region genes. Light chains (LC) are classifiedas either kappa or lambda. Heavy chains (HC) are classified as gamma,mu, alpha, delta, or epsilon, which in turn define the immunoglobulinclasses, IgG, IgM, IgA, IgD and IgE, respectively. A typicalimmunoglobulin (e.g., antibody) structural unit comprises a tetramer.Each tetramer is composed of two identical pairs of polypeptide chains,each pair having one “light” (about 25 kD) and one “heavy” chain (about50-70 kD). The N-terminus of each chain defines a variable region ofabout 100 to 110 or more amino acids primarily responsible for antigenrecognition.

In a full-length antibody, each heavy chain is comprised of a heavychain variable region (abbreviated herein as HCVR or VH) and a heavychain constant region. The heavy chain constant region is comprised ofthree domains, CH1, CH2 and CH3 (and in some instances, CH4). Each lightchain is comprised of a light chain variable region (abbreviated hereinas LCVR or VL) and a light chain constant region. The light chainconstant region is comprised of one domain, C_(L). The VH and VL regionscan be further subdivided into regions of hypervariability, termedcomplementarity determining regions (CDR), interspersed with regionsthat are more conserved, termed framework regions (FR). Each VH and VLis composed of three CDRs and four FRs, arranged from amino-terminus tocarboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3,CDR3, FR4. The extent of the framework region and CDRs has been defined.The sequences of the framework regions of different light or heavychains are relatively conserved within a species, such as humans. Theframework region of an antibody, that is the combined framework regionsof the constituent light and heavy chains, serves to position and alignthe CDRs in three-dimensional space. Immunoglobulin molecules can be ofany type (e.g., IgG, IgE, IgM, IgD, IgA and IgY), class (e.g., IgG1,IgG2, IgG 3, IgG4, IgA1 and IgA2) or subclass.

The CDRs are primarily responsible for binding to an epitope of anantigen. The CDRs of each chain are typically referred to as CDR1, CDR2,CDR3, numbered sequentially starting from the N-terminus, and are alsotypically identified by the chain in which the particular CDR islocated. Thus, a VH CDR3 is located in the variable domain of the heavychain of the antibody in which it is found, whereas a VL CDR1 is theCDR1 from the variable domain of the light chain of the antibody inwhich it is found. Antibodies with different specificities (i.e.different combining sites for different antigens) have different CDRs.Although it is the CDRs that vary from antibody to antibody, only alimited number of amino acid positions within the CDRs are directlyinvolved in antigen binding. These positions within the CDRs are calledspecificity determining residues (SDRs).

The Kabat definition is a standard for numbering the residues in anantibody and is typically used to identify CDR regions. The Kabatdatabase is now maintained online and CDR sequences can be determined,for example, see IMGT/V-QUEST programme version: 3.2.18 Mar. 29, 2011,available on the internet and Brochet, X. et al., Nucl. Acids Res. 36,W503-508, 2008). The Chothia definition is similar to the Kabatdefinition, but the Chothia definition takes into account positions ofcertain structural loop regions. See, e.g., Chothia et al., J. Mol.Biol., 196: 901-17, 1986; Chothia et al., Nature, 342: 877-83, 1989. TheAbM definition uses an integrated suite of computer programs produced byOxford Molecular Group that model antibody structure. See, e.g., Martinet al., Proc. Natl. Acad. Sci. USA, 86:9268-9272, 1989; “AbM™, AComputer Program for Modeling Variable Regions of Antibodies,” Oxford,UK; Oxford Molecular, Ltd. The AbM definition models the tertiarystructure of an antibody from primary sequence using a combination ofknowledge databases and ab initio methods, such as those described bySamudrala et al., “Ab Initio Protein Structure Prediction Using aCombined Hierarchical Approach,” in PROTEINS, Structure, Function andGenetics Suppl., 3:194-198, 1999. The contact definition is based on ananalysis of the available complex crystal structures. See, e.g.,MacCallum et al., J. Mol. Biol., 5:732-45, 1996.

The term “Fc region” is used to define the C-terminal region of animmunoglobulin heavy chain, which may be generated by papain digestionof an intact antibody. The Fc region may be a native sequence Fc regionor a variant Fc region. The Fc region of an immunoglobulin generallycomprises two constant domains, a CH2 domain and a CH3 domain, andoptionally comprises a CH4 domain. The Fc portion of an antibodymediates several important effector functions e.g. cytokine induction,ADCC, phagocytosis, complement dependent cytotoxicity (CDC) andhalf-life/clearance rate of antibody and antigen-antibody complexes(e.g., the neonatal FcR (FcRn) binds to the Fc region of IgG at acidicpH in the endosome and protects IgG from degradation, therebycontributing to the long serum half-life of IgG). Replacements of aminoacid residues in the Fc portion to alter antibody effector function areknown in the art (see, e.g., Winter et al., U.S. Pat. Nos. 5,648,260 and5,624,821).

Antibodies exist as intact immunoglobulins or as a number of wellcharacterized fragments. Such fragments include Fab fragments, Fab′fragments, Fab₂, F(ab)′₂ fragments, single chain Fv proteins (“scFv”)and disulfide stabilized Fv proteins (“dsFv”), that bind to the targetantigen. A scFv protein is a fusion protein in which a light chainvariable region of an immunoglobulin and a heavy chain variable regionof an immunoglobulin are bound by a linker, while in dsFvs, the chainshave been mutated to introduce a disulfide bond to stabilize theassociation of the chains. While various antibody fragments are definedin terms of the digestion of an intact antibody, one of skill willappreciate that such fragments may be synthesized de novo eitherchemically or by utilizing recombinant DNA methodology. Thus, as usedherein, the term antibody encompasses e.g., monoclonal antibodies(including full-length monoclonal antibodies), polyclonal antibodies,multispecific antibodies (e.g., bispecific antibodies) formed from atleast two intact antibodies, human antibodies, humanized antibodies,camelised antibodies, chimeric antibodies, single-chain Fvs (scFv),single-chain antibodies, single domain antibodies, domain antibodies,Fab fragments, F(ab′)₂ fragments, antibody fragments that exhibit thedesired biological activity, disulfide-linked Fvs (sdFv), intrabodies,and epitope-binding fragments or antigen binding fragments of any of theabove.

Papain digestion of antibodies produces two identical antigen-bindingfragments, called “Fab” fragments, each with a single antigen-bindingsite. A “Fab fragment” comprises one light chain and the CH1 andvariable regions of one heavy chain. The heavy chain of a Fab moleculecannot form a disulfide bond with another heavy chain molecule. A “Fab′fragment” comprises one light chain and a portion of one heavy chainthat contains the VH domain and the CH1 domain and also the regionbetween the CH1 and CH2 domains, such that an interchain disulfide bondcan be formed between the two heavy chains of two Fab′ fragments to forman F(ab′)₂ molecule.

Pepsin treatment of an antibody yields an F(ab′)₂ fragment that has twoantigen-combining sites and is still capable of cross-linking antigen. A“F(ab′)₂ fragment” contains two light chains and two heavy chainscontaining a portion of the constant region between the CH1 and CH2domains, such that an interchain disulfide bond is formed between thetwo heavy chains. A F(ab′)₂ fragment thus is composed of two Fab′fragments that are held together by a disulfide bond between the twoheavy chains.

The “Fv region” comprises the variable regions from both the heavy andlight chains, but lacks the constant regions.

“Single-chain antibodies” are Fv molecules in which the heavy and lightchain variable regions have been connected by a flexible linker to forma single polypeptide chain, which forms an antigen binding region.Single chain antibodies are discussed in detail in International PatentApplication Publication No. WO 88/01649, U.S. Pat. Nos. 4,946,778 and5,260,203, the disclosures of which are incorporated by reference.

The terms “an antigen-binding fragment” and “antigen-binding protein” asused herein means any protein that binds a specified target antigen.“Antigen-binding fragment” includes but is not limited to antibodies andbinding parts thereof, such as immunologically functional fragments. Anexemplary antigen-binding fragment of an antibody is the heavy chainand/or light chain CDR(s), or the heavy and/or light chain variableregion.

The term “immunologically functional fragment” (or simply “fragment”) ofan antibody or immunoglobulin chain (heavy or light chain) antigenbinding protein, as used herein, is a species of antigen binding proteincomprising a portion (regardless of how that portion is obtained orsynthesized) of an antibody that lacks at least some of the amino acidspresent in a full-length chain but which is still capable ofspecifically binding to an antigen. Such fragments are biologicallyactive in that they bind to the target antigen and can compete withother antigen binding proteins, including intact antibodies, for bindingto a given epitope. In some embodiments, the fragments are neutralizingfragments. In one aspect, such a fragment will retain at least one CDRpresent in the full-length light or heavy chain, and in some embodimentswill comprise a single heavy chain and/or light chain or portionthereof. These biologically active fragments can be produced byrecombinant DNA techniques, or can be produced by enzymatic or chemicalcleavage of antigen binding proteins, including intact antibodies.Immunologically functional immunoglobulin fragments include, but are notlimited to, Fab, a diabody, Fab′, F(ab′)₂, Fv, domain antibodies andsingle-chain antibodies, and can be derived from any mammalian source,including but not limited to human, mouse, rat, camelid or rabbit. It isfurther contemplated that a functional portion of the antigen bindingproteins disclosed herein, for example, one or more CDRs, could becovalently bound to a second protein or to a small molecule to create atherapeutic agent directed to a particular target in the body,possessing bifunctional therapeutic properties, or having a prolongedserum half-life.

Diabodies are bivalent antibodies comprising two polypeptide chains,wherein each polypeptide chain comprises VH and VL regions joined by alinker that is too short to allow for pairing between two regions on thesame chain, thus allowing each region to pair with a complementaryregion on another polypeptide chain (see, e.g., Holliger et al., Proc.Natl. Acad. Sci. USA, 90:6444-48, 1993; and Poljak et al., Structure,2:1121-23, 1994). If the two polypeptide chains of a diabody areidentical, then a diabody resulting from their pairing will have twoidentical antigen binding sites. Polypeptide chains having differentsequences can be used to make a diabody with two different antigenbinding sites. Similarly, tribodies and tetrabodies are antibodiescomprising three and four polypeptide chains, respectively, and formingthree and four antigen binding sites, respectively, which can be thesame or different.

Bispecific antibodies or fragments can be of several configurations. Forexample, bispecific antibodies may resemble single antibodies (orantibody fragments) but have two different antigen binding sites(variable regions). In various embodiments bispecific antibodies can beproduced by chemical techniques (Kranz et al., Proc. Natl. Acad. Sci.USA, 78:5807, 1981; by “polydoma” techniques (see, e.g., U.S. Pat. No.4,474,893); or by recombinant DNA techniques. In various embodimentsbispecific antibodies of the present disclosure can have bindingspecificities for at least two different epitopes at least one of whichis a tumor associate antigen. In various embodiments the antibodies andfragments can also be heteroantibodies. Heteroantibodies are two or moreantibodies, or antibody binding fragments (e.g., Fab) linked together,each antibody or fragment having a different specificity.

The term “monoclonal antibody” as used herein refers to an antibodyobtained from a population of substantially homogeneous antibodies,i.e., the individual antibodies comprising the population are identicalexcept for possible naturally occurring mutations that may be present inminor amounts. Monoclonal antibodies are highly specific, being directedagainst a single antigen. Furthermore, in contrast to polyclonalantibody preparations that typically include different antibodiesdirected against different determinants (epitopes), each monoclonalantibody is directed against a single determinant on the antigen. Themodifier “monoclonal” is not to be construed as requiring production ofthe antibody by any particular method.

The term “chimeric antibody” as used herein refers to an antibody whichhas framework residues from one species, such as human, and CDRs (whichgenerally confer antigen binding) from another species, such as a murineantibody that specifically binds targeted antigen.

The term “human antibody”, as used herein, is intended to includeantibodies having variable and constant regions derived from humangermline immunoglobulin sequences. The human antibodies of thedisclosure may include amino acid residues not encoded by human germlineimmunoglobulin sequences (e.g., mutations introduced by random orsite-specific mutagenesis in vitro or by somatic mutation in vivo), forexample in the CDRs and in particular CDR3. However, the term “humanantibody”, as used herein, is not intended to include antibodies inwhich CDR sequences derived from the germline of another mammalianspecies, such as a mouse, have been grafted onto human frameworksequences.

The term “humanized antibody” as used herein refers to an antibodycomprising a humanized light chain and a humanized heavy chainimmunoglobulin. A humanized antibody binds to the same antigen as thedonor antibody that provides the CDRs. The acceptor framework of ahumanized immunoglobulin or antibody may have a limited number ofsubstitutions by amino acids taken from the donor framework. Humanizedor other monoclonal antibodies can have additional conservative aminoacid substitutions which have substantially no effect on antigen bindingor other immunoglobulin functions. In various embodiments, the frameworkregions are chosen from human germline exon X_(H), J_(H), V_(K) andJ_(K) sequences. For example, acceptor sequences for humanization of FRof a V_(H) domain can be chosen from genuine V_(H) exons V_(H) 1-18(Matsuda et al., Nature Genetics 3:88-94, 1993) or V_(H)1-2 (Shin etal., EMBO J. 10:3641-3645, 1991) and for the hinge region (J_(H)), exonJ_(H)-6 (Mattila et al., Eur. J. Immunol. 25:2578-2582, 1995). In otherexamples, germline V_(K) exon B3 (Cox et al., Eur. J. Immunol.24:827-836, 1994) and J_(K) exon J_(K)-1 (Hieter et al., J. Biol. Chem.257:1516-1522, 1982) can be chosen as acceptor sequences for V_(L)domain humanization.

The term “recombinant human antibody”, as used herein, is intended toinclude all human antibodies that are prepared, expressed, created orisolated by recombinant means, such as antibodies expressed using arecombinant expression vector transfected into a host cell; antibodiesisolated from a recombinant, combinatorial human antibody library;antibodies isolated from an animal (e.g., a mouse) that is transgenicfor human immunoglobulin genes; or antibodies prepared, expressed,created or isolated by any other means that involves splicing of humanimmunoglobulin gene sequences to other DNA sequences. Such recombinanthuman antibodies have variable and constant regions derived from humangermline immunoglobulin sequences. In various embodiments, however, suchrecombinant human antibodies are subjected to in vitro mutagenesis (or,when an animal transgenic for human Ig sequences is used, in vivosomatic mutagenesis) and thus the amino acid sequences of the VH and VLregions of the recombinant antibodies are sequences that, while derivedfrom and related to human germline VH and VL sequences, may notnaturally exist within the human antibody germline repertoire in vivo.All such recombinant means are well known to those of ordinary skill inthe art.

The term “anti-CGRP antagonist antibody” (interchangeably termed“anti-CGRP antibody”) refers to an antibody that is able to bind to CGRPand inhibit CGRP biological activity and/or downstream pathway(s)mediated by CGRP signaling. An anti-CGRP antagonist antibody encompassesantibodies that block, antagonize, suppress or reduce (includingsignificantly) CGRP biological activity, including downstream pathwaysmediated by CGRP signaling, such as receptor binding and/or elicitationof a cellular response to CGRP. For purpose of the present invention, itwill be explicitly understood that the term “anti-CGRP antagonistantibody” encompasses all the previously identified terms, titles, andfunctional states and characteristics whereby the CGRP itself, an CGRPbiological activity (including but not limited to its ability to mediateany aspect of headache), or the consequences of the biological activity,are substantially nullified, decreased, or neutralized in any meaningfuldegree. In some embodiment, an anti-CGRP antagonist antibody binds CGRPand prevents CGRP binding to a CGRP receptor. In other embodiments, ananti-CGRP antibody binds CGRP and prevents activation of a CGRPreceptor. Examples of anti-CGRP antagonist antibodies are providedherein.

The term “epitope” as used herein includes any protein determinantcapable of specific binding to an immunoglobulin or T-cell receptor orotherwise interacting with a molecule. Epitopic determinants generallyconsist of chemically active surface groupings of molecules such asamino acids or carbohydrate or sugar side chains and generally havespecific three dimensional structural characteristics, as well asspecific charge characteristics. An epitope may be “linear” or“conformational.” In a linear epitope, all of the points of interactionbetween the protein and the interacting molecule (such as an antibody)occur linearly along the primary amino acid sequence of the protein. Ina conformational epitope, the points of interaction occur across aminoacid residues on the protein that are separated from one another. Once adesired epitope on an antigen is determined, it is possible to generateantibodies to that epitope, e.g., using the techniques described in thepresent disclosure. Alternatively, during the discovery process, thegeneration and characterization of antibodies may elucidate informationabout desirable epitopes. From this information, it is then possible tocompetitively screen antibodies for binding to the same epitope. Anapproach to achieve this is to conduct cross-competition studies to findantibodies that competitively bind with one another, e.g., theantibodies compete for binding to the antigen.

An antigen binding protein, including an antibody, “specifically binds”to an antigen if it binds to the antigen with a high binding affinity asdetermined by a dissociation constant (K_(D), or corresponding Kb, asdefined below) value of at least 1×10⁻⁶ M, or at least 1×10⁻⁷ M, or atleast 1×10⁻⁸ M, or at least 1×10⁻⁹ M, or at least 1×10⁻¹⁰ M, or at least1×10⁻¹¹ M. An antigen binding protein that specifically binds to thehuman antigen of interest may be able to bind to the same antigen ofinterest from other species as well, with the same or differentaffinities. The term “K_(D)” as used herein refers to the equilibriumdissociation constant of a particular antibody-antigen interaction.

The term “surface plasmon resonance” as used herein refers to an opticalphenomenon that allows for the analysis of real-time biospecificinteractions by detection of alterations in protein concentrationswithin a biosensor matrix, for example using the BIACORE™ system(Pharmacia Biosensor AB, Uppsala, Sweden and Piscataway, N.J.). Forfurther descriptions, see Jonsson U. et al., Ann. Biol. Clin., 51:19-26,1993; Jonsson U. et al., Biotechniques, 11:620-627, 1991; Jonsson B. etal., J. Mol. Recognit., 8:125-131, 1995; and Johnsson B. et al., Anal.Biochem, 198:268-277, 1991.

The term “immunogenicity” as used herein refers to the ability of anantibody or antigen binding fragment to elicit an immune response(humoral or cellular) when administered to a recipient and includes, forexample, the human anti-mouse antibody (HAMA) response. A HAMA responseis initiated when T-cells from a subject make an immune response to theadministered antibody. The T-cells then recruit B-cells to generatespecific “anti-antibody” antibodies.

The term “immune cell” as used herein means any cell of hematopoieticlineage involved in regulating an immune response against an antigen(e.g., an autoantigen). In various embodiments, an immune cell is, e.g.,a T cell, a B cell, a dendritic cell, a monocyte, a natural killer cell,a macrophage, Langerhan's cells, or Kuffer cells.

The terms “polypeptide”, “peptide” and “protein” are usedinterchangeably herein to refer to a polymer of amino acid residues. Invarious embodiments, “peptides”, “polypeptides”, and “proteins” arechains of amino acids whose alpha carbons are linked through peptidebonds. The terminal amino acid at one end of the chain (amino terminal)therefore has a free amino group, while the terminal amino acid at theother end of the chain (carboxy terminal) has a free carboxyl group. Asused herein, the term “amino terminus” (abbreviated N-terminus) refersto the free α-amino group on an amino acid at the amino terminal of apeptide or to the α-amino group (imino group when participating in apeptide bond) of an amino acid at any other location within the peptide.Similarly, the term “carboxy terminus” refers to the free carboxyl groupon the carboxy terminus of a peptide or the carboxyl group of an aminoacid at any other location within the peptide. Peptides also includeessentially any polyamino acid including, but not limited to, peptidemimetics such as amino acids joined by an ether as opposed to an amidebond.

The term “recombinant polypeptide”, as used herein, is intended toinclude all polypeptides, including fusion molecules that are prepared,expressed, created, derived from, or isolated by recombinant means, suchas polypeptides expressed using a recombinant expression vectortransfected into a host cell.

Polypeptides of the disclosure include polypeptides that have beenmodified in any way and for any reason, for example, to: (1) reducesusceptibility to proteolysis, (2) reduce susceptibility to oxidation,(3) alter binding affinity for forming protein complexes, (4) alterbinding affinities, and (5) confer or modify other physicochemical orfunctional properties. For example, single or multiple amino acidsubstitutions (e.g., conservative amino acid substitutions) may be madein the naturally occurring sequence (e.g., in the portion of thepolypeptide outside the domain(s) forming intermolecular contacts). A“conservative amino acid substitution” refers to the substitution in apolypeptide of an amino acid with a functionally similar amino acid. Thefollowing six groups each contain amino acids that are conservativesubstitutions for one another:

Alanine (A), Serine (S), and Threonine (T)

Aspartic acid (D) and Glutamic acid (E)

Asparagine (N) and Glutamine (Q)

Arginine (R) and Lysine (K)

Isoleucine (I), Leucine (L), Methionine (M), and Valine (V)

Phenylalanine (F), Tyrosine (Y), and Tryptophan (W)

A “non-conservative amino acid substitution” refers to the substitutionof a member of one of these classes for a member from another class. Inmaking such changes, according to various embodiments, the hydropathicindex of amino acids may be considered. Each amino acid has beenassigned a hydropathic index on the basis of its hydrophobicity andcharge characteristics. They are: isoleucine (+4.5); valine (+4.2);leucine (+3.8); phenylalanine (+2.8); cysteine/cystine (+2.5);methionine (+1.9); alanine (+1.8); glycine (−0.4); threonine (−0.7);serine (−0.8); tryptophan (−0.9); tyrosine (−1.3); proline (−1.6);histidine (−3.2); glutamate (−3.5); glutamine (−3.5); aspartate (−3.5);asparagine (−3.5); lysine (−3.9); and arginine (−4.5).

The importance of the hydropathic amino acid index in conferringinteractive biological function on a protein is understood in the art(see, for example, Kyte et al., 1982, J. Mol. Biol. 157:105-131). It isknown that certain amino acids may be substituted for other amino acidshaving a similar hydropathic index or score and still retain a similarbiological activity. In making changes based upon the hydropathic index,in various embodiments, the substitution of amino acids whosehydropathic indices are within ±2 is included. In various embodiments,those that are within ±1 are included, and in various embodiments, thosewithin ±0.5 are included.

It is also understood in the art that the substitution of like aminoacids can be made effectively on the basis of hydrophilicity,particularly where the biologically functional protein or peptidethereby created is intended for use in immunological embodiments, asdisclosed herein. In various embodiments, the greatest local averagehydrophilicity of a protein, as governed by the hydrophilicity of itsadjacent amino acids, correlates with its immunogenicity andantigenicity, i.e., with a biological property of the protein.

The following hydrophilicity values have been assigned to these aminoacid residues: arginine (+3.0); lysine (+3.0); aspartate (+3.0.+−0.1);glutamate (+3.0.+−0.1); serine (+0.3); asparagine (+0.2); glutamine(+0.2); glycine (0); threonine (−0.4); proline (−0.5.+−0.1); alanine(−0.5); histidine (−0.5); cysteine (−1.0); methionine (−1.3); valine(−1.5); leucine (−1.8); isoleucine (−1.8); tyrosine (−2.3);phenylalanine (−2.5) and tryptophan (−3.4). In making changes based uponsimilar hydrophilicity values, in various embodiments, the substitutionof amino acids whose hydrophilicity values are within ±2 is included, invarious embodiments, those that are within ±1 are included, and invarious embodiments, those within ±0.5 are included. Exemplary aminoacid substitutions are set forth in Table 1.

TABLE 1 Original Exemplary Preferred Residues SubstitutionsSubstitutions Ala Val, Leu, Ile Val Arg Lys, Gln, Asn Lys Asn Gln AspGlu Cys Ser, Ala Ser Gln Asn Asn Glu Asp Asp Gly Pro, Ala Ala His Asn,Gln, Lys, Arg Arg Ile Leu, Val, Met, Ala, Leu Phe, Norleucine LeuNorleucine, Ile, Ile Val, Met, Ala, Phe Lys Arg, 1,4 Diamino-butyric ArgAcid, Gln, Asn Met Leu, Phe, Ile Leu Phe Leu, Val, Ile, Ala, Tyr Leu ProAla Gly Ser Thr, Ala, Cys Thr Thr Ser Trp Tyr, Phe Tyr Tyr Trp, Phe,Thr, Ser Phe Val Ile, Met, Leu, Phe, Leu Ala, Norleucine

The term “polypeptide fragment” and “truncated polypeptide” as usedherein refers to a polypeptide that has an amino-terminal and/orcarboxy-terminal deletion as compared to a corresponding full-lengthprotein. In various embodiments, fragments can be, e.g., at least 5, atleast 10, at least 25, at least 50, at least 100, at least 150, at least200, at least 250, at least 300, at least 350, at least 400, at least450, at least 500, at least 600, at least 700, at least 800, at least900 or at least 1000 amino acids in length. In various embodiments,fragments can also be, e.g., at most 1000, at most 900, at most 800, atmost 700, at most 600, at most 500, at most 450, at most 400, at most350, at most 300, at most 250, at most 200, at most 150, at most 100, atmost 50, at most 25, at most 10, or at most 5 amino acids in length. Afragment can further comprise, at either or both of its ends, one ormore additional amino acids, for example, a sequence of amino acids froma different naturally-occurring protein (e.g., an Fc or leucine zipperdomain) or an artificial amino acid sequence (e.g., an artificial linkersequence).

The terms “polypeptide variant” and “polypeptide mutant” as used hereinrefers to a polypeptide that comprises an amino acid sequence whereinone or more amino acid residues are inserted into, deleted from and/orsubstituted into the amino acid sequence relative to another polypeptidesequence. In various embodiments, the number of amino acid residues tobe inserted, deleted, or substituted can be, e.g., at least 1, at least2, at least 3, at least 4, at least 5, at least 10, at least 25, atleast 50, at least 75, at least 100, at least 125, at least 150, atleast 175, at least 200, at least 225, at least 250, at least 275, atleast 300, at least 350, at least 400, at least 450 or at least 500amino acids in length. Variants of the present disclosure include fusionproteins.

A “derivative” of a polypeptide is a polypeptide that has beenchemically modified, e.g., conjugation to another chemical moiety suchas, for example, polyethylene glycol, albumin (e.g., human serumalbumin), phosphorylation, and glycosylation.

The term “% sequence identity” is used interchangeably herein with theterm “% identity” and refers to the level of amino acid sequenceidentity between two or more peptide sequences or the level ofnucleotide sequence identity between two or more nucleotide sequences,when aligned using a sequence alignment program. For example, as usedherein, 80% identity means the same thing as 80% sequence identitydetermined by a defined algorithm, and means that a given sequence is atleast 80% identical to another length of another sequence. In variousembodiments, the % identity is selected from, e.g., at least 60%, atleast 65%, at least 70%, at least 75%, at least 80%, at least 85%, atleast 90%, at least 95%, or at least 99% or more sequence identity to agiven sequence. In various embodiments, the % identity is in the rangeof, e.g., about 60% to about 70%, about 70% to about 80%, about 80% toabout 85%, about 85% to about 90%, about 90% to about 95%, or about 95%to about 99%.

The term “% sequence homology” is used interchangeably herein with theterm “% homology” and refers to the level of amino acid sequencehomology between two or more peptide sequences or the level ofnucleotide sequence homology between two or more nucleotide sequences,when aligned using a sequence alignment program. For example, as usedherein, 80% homology means the same thing as 80% sequence homologydetermined by a defined algorithm, and accordingly a homologue of agiven sequence has greater than 80% sequence homology over a length ofthe given sequence. In various embodiments, the % homology is selectedfrom, e.g., at least 60%, at least 65%, at least 70%, at least 75%, atleast 80%, at least 85%, at least 90%, at least 95%, or at least 99% ormore sequence homology to a given sequence. In various embodiments, the% homology is in the range of, e.g., about 60% to about 70%, about 70%to about 80%, about 80% to about 85%, about 85% to about 90%, about 90%to about 95%, or about 95% to about 99%.

Exemplary computer programs which can be used to determine identitybetween two sequences include, but are not limited to, the suite ofBLAST programs, e.g., BLASTN, BLASTX, and TBLASTX, BLASTP and TBLASTN,publicly available on the Internet at the NCBI website. See alsoAltschul et al., J. Mol. Biol. 215:403-10, 1990 (with special referenceto the published default setting, i.e., parameters w=4, t=17) andAltschul et al., Nucleic Acids Res., 25:3389-3402, 1997. Sequencesearches are typically carried out using the BLASTP program whenevaluating a given amino acid sequence relative to amino acid sequencesin the GenBank Protein Sequences and other public databases. The BLASTXprogram is preferred for searching nucleic acid sequences that have beentranslated in all reading frames against amino acid sequences in theGenBank Protein Sequences and other public databases. Both BLASTP andBLASTX are run using default parameters of an open gap penalty of 11.0,and an extended gap penalty of 1.0, and utilize the BLOSUM-62 matrix.See Id.

In addition to calculating percent sequence identity, the BLASTalgorithm also performs a statistical analysis of the similarity betweentwo sequences (see, e.g., Karlin & Altschul, Proc. Nat'l. Acad. Sci.USA, 90:5873-5787, 1993). One measure of similarity provided by theBLAST algorithm is the smallest sum probability (P(N)), which providesan indication of the probability by which a match between two nucleotideor amino acid sequences would occur by chance. For example, a nucleicacid is considered similar to a reference sequence if the smallest sumprobability in a comparison of the test nucleic acid to the referencenucleic acid is, e.g., less than about 0.1, less than about 0.01, orless than about 0.001.

The terms “substantial similarity” or “substantially similar,” in thecontext of polypeptide sequences, indicate that a polypeptide region hasa sequence with at least 70%, typically at least 80%, more typically atleast 85%, or at least 90% or at least 95% sequence similarity to areference sequence. For example, a polypeptide is substantially similarto a second polypeptide, for example, where the two peptides differ byone or more conservative substitution(s).

“Polynucleotide” refers to a polymer composed of nucleotide units.Polynucleotides include naturally occurring nucleic acids, such asdeoxyribonucleic acid (“DNA”) and ribonucleic acid (“RNA”) as well asnucleic acid analogs. Nucleic acid analogs include those which includenon-naturally occurring bases, nucleotides that engage in linkages withother nucleotides other than the naturally occurring phosphodiester bondor which include bases attached through linkages other thanphosphodiester bonds. Thus, nucleotide analogs include, for example andwithout limitation, phosphorothioates, phosphorodithioates,phosphorotriesters, phosphoramidates, boranophosphates,methylphosphonates, chiral-methyl phosphonates, 2-O-methylribonucleotides, peptide-nucleic acids (PNAs), and the like. Suchpolynucleotides can be synthesized, for example, using an automated DNAsynthesizer. The term “nucleic acid” typically refers to largepolynucleotides. The term “oligonucleotide” typically refers to shortpolynucleotides, generally no greater than about 50 nucleotides. It willbe understood that when a nucleotide sequence is represented by a DNAsequence (i.e., A, T, G, C), this also includes an RNA sequence (i.e.,A, U, G, C) in which “U” replaces “T.”

Conventional notation is used herein to describe polynucleotidesequences: the left-hand end of a single-stranded polynucleotidesequence is the 5′-end; the left-hand direction of a double-strandedpolynucleotide sequence is referred to as the 5′-direction. Thedirection of 5′ to 3′ addition of nucleotides to nascent RNA transcriptsis referred to as the transcription direction. The DNA strand having thesame sequence as an mRNA is referred to as the “coding strand”;sequences on the DNA strand having the same sequence as an mRNAtranscribed from that DNA and which are located 5′ to the 5′-end of theRNA transcript are referred to as “upstream sequences”; sequences on theDNA strand having the same sequence as the RNA and which are 3′ to the3′ end of the coding RNA transcript are referred to as “downstreamsequences.”

“Complementary” refers to the topological compatibility or matchingtogether of interacting surfaces of two polynucleotides. Thus, the twomolecules can be described as complementary, and furthermore, thecontact surface characteristics are complementary to each other. A firstpolynucleotide is complementary to a second polynucleotide if thenucleotide sequence of the first polynucleotide is substantiallyidentical to the nucleotide sequence of the polynucleotide bindingpartner of the second polynucleotide, or if the first polynucleotide canhybridize to the second polynucleotide under stringent hybridizationconditions.

“Hybridizing specifically to” or “specific hybridization” or“selectively hybridize to”, refers to the binding, duplexing, orhybridizing of a nucleic acid molecule preferentially to a particularnucleotide sequence under stringent conditions when that sequence ispresent in a complex mixture (e.g., total cellular) DNA or RNA. The term“stringent conditions” refers to conditions under which a probe willhybridize preferentially to its target subsequence, and to a lesserextent to, or not at all to, other sequences. “Stringent hybridization”and “stringent hybridization wash conditions” in the context of nucleicacid hybridization experiments such as Southern and northernhybridizations are sequence-dependent, and are different under differentenvironmental parameters. An extensive guide to the hybridization ofnucleic acids can be found in Tijssen, 1993, Laboratory Techniques inBiochemistry and Molecular Biology—Hybridization with Nucleic AcidProbes, part I, chapter 2, “Overview of principles of hybridization andthe strategy of nucleic acid probe assays”, Elsevier, N.Y.; Sambrook etal., 2001, Molecular Cloning: A Laboratory Manual, Cold Spring HarborLaboratory, 3.sup.rd ed., NY; and Ausubel et al., eds., Current Edition,Current Protocols in Molecular Biology, Greene Publishing Associates andWiley Interscience, NY.

Generally, highly stringent hybridization and wash conditions areselected to be about 5° C. lower than the thermal melting point (Tm) forthe specific sequence at a defined ionic strength and pH. The Tm is thetemperature (under defined ionic strength and pH) at which 50% of thetarget sequence hybridizes to a perfectly matched probe. Very stringentconditions are selected to be equal to the Tm for a particular probe. Anexample of stringent hybridization conditions for hybridization ofcomplementary nucleic acids which have more than about 100 complementaryresidues on a filter in a Southern or northern blot is 50% formalin with1 mg of heparin at 42° C., with the hybridization being carried outovernight. An example of highly stringent wash conditions is 0.15 M NaClat 72° C. for about 15 minutes. An example of stringent wash conditionsis a 0.2×SSC wash at 65° C. for 15 minutes. See Sambrook et al. for adescription of SSC buffer. A high stringency wash can be preceded by alow stringency wash to remove background probe signal. An exemplarymedium stringency wash for a duplex of, e.g., more than about 100nucleotides, is 1×SSC at 45° C. for 15 minutes. An exemplary lowstringency wash for a duplex of, e.g., more than about 100 nucleotides,is 4-6×SSC at 40° C. for 15 minutes. In general, a signal to noise ratioof 2× (or higher) than that observed for an unrelated probe in theparticular hybridization assay indicates detection of a specifichybridization.

“Primer” refers to a polynucleotide that is capable of specificallyhybridizing to a designated polynucleotide template and providing apoint of initiation for synthesis of a complementary polynucleotide.Such synthesis occurs when the polynucleotide primer is placed underconditions in which synthesis is induced, i.e., in the presence ofnucleotides, a complementary polynucleotide template, and an agent forpolymerization such as DNA polymerase. A primer is typicallysingle-stranded, but may be double-stranded. Primers are typicallydeoxyribonucleic acids, but a wide variety of synthetic and naturallyoccurring primers are useful for many applications. A primer iscomplementary to the template to which it is designed to hybridize toserve as a site for the initiation of synthesis, but need not reflectthe exact sequence of the template. In such a case, specifichybridization of the primer to the template depends on the stringency ofthe hybridization conditions. Primers can be labeled with, e.g.,chromogenic, radioactive, or fluorescent moieties and used as detectablemoieties.

“Probe,” when used in reference to a polynucleotide, refers to apolynucleotide that is capable of specifically hybridizing to adesignated sequence of another polynucleotide. A probe specificallyhybridizes to a target complementary polynucleotide, but need notreflect the exact complementary sequence of the template. In such acase, specific hybridization of the probe to the target depends on thestringency of the hybridization conditions. Probes can be labeled with,e.g., chromogenic, radioactive, or fluorescent moieties and used asdetectable moieties. In instances where a probe provides a point ofinitiation for synthesis of a complementary polynucleotide, a probe canalso be a primer.

A “vector” is a polynucleotide that can be used to introduce anothernucleic acid linked to it into a cell. One type of vector is a“plasmid,” which refers to a linear or circular double stranded DNAmolecule into which additional nucleic acid segments can be ligated.Another type of vector is a viral vector (e.g., replication defectiveretroviruses, adenoviruses and adeno-associated viruses), whereinadditional DNA segments can be introduced into the viral genome. Certainvectors are capable of autonomous replication in a host cell into whichthey are introduced (e.g., bacterial vectors comprising a bacterialorigin of replication and episomal mammalian vectors). Other vectors(e.g., non-episomal mammalian vectors) are integrated into the genome ofa host cell upon introduction into the host cell, and thereby arereplicated along with the host genome. An “expression vector” is a typeof vector that can direct the expression of a chosen polynucleotide.

A “regulatory sequence” is a nucleic acid that affects the expression(e.g., the level, timing, or location of expression) of a nucleic acidto which it is operably linked. The regulatory sequence can, forexample, exert its effects directly on the regulated nucleic acid, orthrough the action of one or more other molecules (e.g., polypeptidesthat bind to the regulatory sequence and/or the nucleic acid). Examplesof regulatory sequences include promoters, enhancers and otherexpression control elements (e.g., polyadenylation signals). Furtherexamples of regulatory sequences are described in, for example, Goeddel,1990, Gene Expression Technology: Methods in Enzymology 185, AcademicPress, San Diego, Calif. and Baron et al., 1995, Nucleic Acids Res.23:3605-06. A nucleotide sequence is “operably linked” to a regulatorysequence if the regulatory sequence affects the expression (e.g., thelevel, timing, or location of expression) of the nucleotide sequence.

A “host cell” is a cell that can be used to express a polynucleotide ofthe disclosure. A host cell can be a prokaryote, for example, E. coli,or it can be a eukaryote, for example, a single-celled eukaryote (e.g.,a yeast or other fungus), a plant cell (e.g., a tobacco or tomato plantcell), an animal cell (e.g., a human cell, a monkey cell, a hamstercell, a rat cell, a mouse cell, or an insect cell) or a hybridoma.Typically, a host cell is a cultured cell that can be transformed ortransfected with a polypeptide-encoding nucleic acid, which can then beexpressed in the host cell. The phrase “recombinant host cell” can beused to denote a host cell that has been transformed or transfected witha nucleic acid to be expressed. A host cell also can be a cell thatcomprises the nucleic acid but does not express it at a desired levelunless a regulatory sequence is introduced into the host cell such thatit becomes operably linked with the nucleic acid. It is understood thatthe term host cell refers not only to the particular subject cell but tothe progeny or potential progeny of such a cell. Because certainmodifications may occur in succeeding generations due to, e.g., mutationor environmental influence, such progeny may not, in fact, be identicalto the parent cell, but are still included within the scope of the termas used herein.

The term “isolated molecule” (where the molecule is, for example, apolypeptide or a polynucleotide) is a molecule that by virtue of itsorigin or source of derivation (1) is not associated with naturallyassociated components that accompany it in its native state, (2) issubstantially free of other molecules from the same species (3) isexpressed by a cell from a different species, or (4) does not occur innature. Thus, a molecule that is chemically synthesized, or expressed ina cellular system different from the cell from which it naturallyoriginates, will be “isolated” from its naturally associated components.A molecule also may be rendered substantially free of naturallyassociated components by isolation, using purification techniques wellknown in the art. Molecule purity or homogeneity may be assayed by anumber of means well known in the art. For example, the purity of apolypeptide sample may be assayed using polyacrylamide gelelectrophoresis and staining of the gel to visualize the polypeptideusing techniques well known in the art. For certain purposes, higherresolution may be provided by using HPLC or other means well known inthe art for purification.

A protein or polypeptide is “substantially pure,” “substantiallyhomogeneous,” or “substantially purified” when at least about 60% to 75%of a sample exhibits a single species of polypeptide. The polypeptide orprotein may be monomeric or multimeric. A substantially pure polypeptideor protein will typically comprise about 50%, 60%, 70%, 80% or 90% W/Wof a protein sample, more usually about 95%, and preferably will be over99% pure. Protein purity or homogeneity may be indicated by a number ofmeans well known in the art, such as polyacrylamide gel electrophoresisof a protein sample, followed by visualizing a single polypeptide bandupon staining the gel with a stain well known in the art. For certainpurposes, higher resolution may be provided by using HPLC or other meanswell known in the art for purification.

“Linker” refers to a molecule that joins two other molecules, eithercovalently, or through ionic, van der Waals or hydrogen bonds, e.g., anucleic acid molecule that hybridizes to one complementary sequence atthe 5′ end and to another complementary sequence at the 3′ end, thusjoining two non-complementary sequences. A “cleavable linker” refers toa linker that can be degraded or otherwise severed to separate the twocomponents connected by the cleavable linker. Cleavable linkers aregenerally cleaved by enzymes, typically peptidases, proteases,nucleases, lipases, and the like. Cleavable linkers may also be cleavedby environmental cues, such as, for example, changes in temperature, pH,salt concentration, etc.

The terms “label” or “labeled” as used herein refers to incorporation ofanother molecule in the antibody. In one embodiment, the label is adetectable marker, e.g., incorporation of a radiolabeled amino acid orattachment to a polypeptide of biotinyl moieties that can be detected bymarked avidin (e.g., streptavidin containing a fluorescent marker orenzymatic activity that can be detected by optical or calorimetricmethods). In another embodiment, the label or marker can be therapeutic,e.g., a drug conjugate or toxin. Various methods of labelingpolypeptides and glycoproteins are known in the art and may be used.Examples of labels for polypeptides include, but are not limited to, thefollowing: radioisotopes or radionuclides (e.g., ³H, ¹⁴C, ¹⁵N, ³⁵S, ⁹⁰Y,⁹⁹Tc, ¹¹¹In, ¹²⁵I, ¹³¹I), fluorescent labels (e.g., FITC, rhodamine,lanthanide phosphors), enzymatic labels (e.g., horseradish peroxidase,β-galactosidase, luciferase, alkaline phosphatase), chemiluminescentmarkers, biotinyl groups, predetermined polypeptide epitopes recognizedby a secondary reporter (e.g., leucine zipper pair sequences, bindingsites for secondary antibodies, metal binding domains, epitope tags),magnetic agents, such as gadolinium chelates, toxins such as pertussistoxin, taxol, cytochalasin B, gramicidin D, ethidium bromide, emetine,mitomycin, etoposide, tenoposide, vincristine, vinblastine, colchicine,doxorubicin, daunorubicin, dihydroxy anthracin dione, mitoxantrone,mithramycin, actinomycin D, 1-dehydrotestosterone, glucocorticoids,procaine, tetracaine, lidocaine, propranolol, and puromycin and analogsor homologs thereof. In some embodiments, labels are attached by spacerarms of various lengths to reduce potential steric hindrance.

The term “immunoconjugate” or “fusion protein” as used herein refers toa molecule comprising an antibody or antigen-binding fragment thereofconjugated (or linked) directly or indirectly to an effector molecule.The effector molecule can be a detectable label, an immunotoxin,cytokine, chemokine, therapeutic agent, or chemotherapeutic agent. Theantibody or antigen-binding fragment thereof may be conjugated to aneffector molecule via a peptide linker. An immunoconjugate and/or fusionprotein retains the immunoreactivity of the antibody or antigen-bindingfragment, e.g., the antibody or antigen-binding fragment hasapproximately the same, or only slightly reduced, ability to bind theantigen after conjugation as before conjugation. As used herein, animmunoconjugate may also be referred to as an antibody drug conjugate(ADC). Because immunoconjugates and/or fusion proteins are originallyprepared from two molecules with separate functionalities, such as anantibody and an effector molecule, they are also sometimes referred toas “chimeric molecules.”

“Pharmaceutical composition” refers to a composition suitable forpharmaceutical use in an animal. A pharmaceutical composition comprisesa pharmacologically effective amount of an active agent and apharmaceutically acceptable carrier. “Pharmacologically effectiveamount” refers to that amount of an agent effective to produce theintended pharmacological result. “Pharmaceutically acceptable carrier”refers to any of the standard pharmaceutical carriers, vehicles,buffers, and excipients, such as a phosphate buffered saline solution,5% aqueous solution of dextrose, and emulsions, such as an oil/water orwater/oil emulsion, and various types of wetting agents and/oradjuvants. Suitable pharmaceutical carriers and formulations aredescribed in Remington's Pharmaceutical Sciences, 21st Ed. 2005, MackPublishing Co, Easton. A “pharmaceutically acceptable salt” is a saltthat can be formulated into a compound for pharmaceutical use including,e.g., metal salts (sodium, potassium, magnesium, calcium, etc.) andsalts of ammonia or organic amines.

The terms “treat”, “treating” and “treatment” refer to a method ofalleviating or abrogating a biological disorder and/or at least one ofits attendant symptoms. As used herein, to “alleviate” a disease,disorder or condition means reducing the severity and/or occurrencefrequency of the symptoms of the disease, disorder, or condition. Asused herein, “treatment” is an approach for obtaining beneficial ordesired clinical results. For purposes of this invention, beneficial ordesired clinical results include, but are not limited to, any one ormore of: alleviation of one or more symptoms, diminishment of extent ofdisease, preventing or delaying spread (e.g., metastasis, for examplemetastasis to the lung or to the lymph node) of disease, preventing ordelaying recurrence of disease, delay or slowing of disease progression,amelioration of the disease state, and remission (whether partial ortotal). Also encompassed by “treatment” is a reduction of pathologicalconsequence of a proliferative disease. The methods of the inventioncontemplate any one or more of these aspects of treatment.

The term “effective amount” or “therapeutically effective amount” asused herein refers to an amount of a compound or composition sufficientto treat a specified disorder, condition or disease such as ameliorate,palliate, lessen, and/or delay one or more of its symptoms.

It is understood that aspects and embodiments of the invention describedherein include “consisting” and/or “consisting essentially of” aspectsand embodiments.

Reference to “about” a value or parameter herein includes (anddescribes) variations that are directed to that value or parameter perse. For example, description referring to “about X” includes descriptionof “X”.

As used herein and in the appended claims, the singular forms “a,” “or,”and “the” include plural referents unless the context clearly dictatesotherwise. It is understood that aspects and variations of the inventiondescribed herein include “consisting” and/or “consisting essentially of”aspects and variations.

CGRP

Calcitonin Gene-related Peptide (CGRP) is a 37 amino acid neuropeptide,which belongs to a family of peptides that includes calcitonin,adrenomedullin and amylin. In humans, two forms of CGRP (α-CGRP andβ-CGRP) exist and have similar activities. They vary by three aminoacids and exhibit differential distribution. At least two CGRP receptorsubtypes may also account for differential activities. CGRP has beendetected in nerves innervating the heart, peripheral and cerebral bloodvessels, and kidneys by immunohistochemical (such as ELISA) andradioimmunoassay methods. CGRP has been shown to mediate its biologicalresponse by binding to specific cell surface receptors that have beenidentified in a variety of tissues. CGRP is a potent vasodilator thathas been implicated in the pathology of other vasomotor symptoms, suchas all forms of vascular headache, including migraines (with or withoutaura) and cluster headache (Durham, N. Engl. J. Med. 350:1073-1075,2004). CGRP has also been noted for its possible connection to vasomotorsymptoms (VMS), such as hot flushes and night sweats (Wyon et al. Scand.J. Urol. Nephrol. 35: 92-96 (2001); Wyon et al. Menopause 7(1):25-30,2000). CGRP is the most prominent neuropeptide in the skin and is alsofound in other tissues, e.g. bone and joints. CGRP stimulateskeratinocyte, i.e., epidermal cell, proliferation (Takahashi et al., JInvest Dermatol, 101(5):646-651, 1993). CGRP is a very importantneuropeptide (NP) in wound healing and is the first NP that is releasedduring that process. CGRP is a very strong vasodilator and is a stronginhibitor of delayed type hypersensitivity (DTH).

The term “CGRP” as used herein includes human CGRP (hCGRP), variants,isoforms, and species homologs of hCGRP, and analogs having at least onecommon epitope with hCGRP. In various embodiments, a hCGRP as usedherein may comprise the amino acid sequence set forth in SEQ ID NO: 1:

(SEQ ID NO: 1) ACDTATCVTHRLAGLLSRSGGVVKNNFVPTNVGSKAF

In various embodiments, a CGRP comprises an amino acid sequence thatshares an observed homology of, e.g., at least 70%, at least 75%, atleast 80%, at least 85%, at least 90%, at least 95%, at least 96%, atleast 97%, at least 98%, or at least 99% with the human CGRP sequence ofSEQ ID NO: 1. In some embodiments, the has at least 70%, at least 75%,at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, atleast 97%, at least 98%, at least 99%, at least 1×, at least 1.5×, atleast 2×, at least 2.5×, or at least 3× activity of the human CGRP ofSEQ ID NO: 1. Variants of CGRP may be described herein by reference tothe addition, deletion, or substitution of amino acid residue present ata given position in the 223 amino acid sequence of SEQ ID NO: 1. Thus,for example, the term “P29W” indicates that the “P” (proline, instandard single letter code) residue at position 29 in SEQ ID NO: 1 hasbeen substituted with a “W” (tryptophan, in standard single lettercode).

Antibodies

Methods of generating novel antibodies that bind to human CGRP are knownto those skilled in the art. For example, a method for generating amonoclonal antibody that binds specifically to an CGRP may compriseadministering to a mouse an amount of an immunogenic compositioncomprising the CGRP effective to stimulate a detectable immune response,obtaining antibody-producing cells (e.g., cells from the spleen) fromthe mouse and fusing the antibody-producing cells with myeloma cells toobtain antibody-producing hybridomas, and testing the antibody-producinghybridomas to identify a hybridoma that produces a monoclonal antibodythat binds specifically to the CGRP. Once obtained, a hybridoma can bepropagated in a cell culture, optionally in culture conditions where thehybridoma-derived cells produce the monoclonal antibody that bindsspecifically to CGRP. The monoclonal antibody may be purified from thecell culture. A variety of different techniques are then available fortesting antibody:antigen interactions to identify particularly desirableantibodies.

Other suitable methods of producing or isolating antibodies of therequisite specificity can used, including, for example, methods whichselect recombinant antibody from a library, or which rely uponimmunization of transgenic animals (e.g., mice) capable of producing afull repertoire of human antibodies. See e.g., Jakobovits et al., Proc.Natl. Acad. Sci. USA, 90: 2551-2555, 1993; Jakobovits et al., Nature,362:255-258, 1993; Lonberg et al., U.S. Pat. No. 5,545,806; Surani etal., U.S. Pat. No. 5,545,807.

Antibodies can be engineered in numerous ways. They can be made assingle-chain antibodies (including small modular immunopharmaceuticalsor SMIPs™), Fab and F(ab′)₂ fragments, etc. Antibodies can be humanized,chimerized, deimmunized, or fully human. Numerous publications set forththe many types of antibodies and the methods of engineering suchantibodies. For example, see U.S. Pat. Nos. 6,355,245; 6,180,370;5,693,762; 6,407,213; 6,548,640; 5,565,332; 5,225,539; 6,103,889; and5,260,203.

Chimeric antibodies can be produced by recombinant DNA techniques knownin the art. For example, a gene encoding the Fc constant region of amurine (or other species) monoclonal antibody molecule is digested withrestriction enzymes to remove the region encoding the murine Fc, and theequivalent portion of a gene encoding a human Fc constant region issubstituted (see Robinson et al., International Patent PublicationPCT/US86/02269; Akira, et al., European Patent Application 184,187;Taniguchi, M., European Patent Application 171,496; Morrison et al.,European Patent Application 173,494; Neuberger et al., InternationalApplication WO 86/01533; Cabilly et al. U.S. Pat. No. 4,816,567; Cabillyet al., European Patent Application 125,023; Better et al., Science,240:1041-1043, 1988; Liu et al., PNAS USA, 84:3439-3443, 1987; Liu etal., J. Immunol. 139:3521-3526, 1987; Sun et al., PNAS USA, 84:214-218,1987; Nishimura et al., Canc. Res. 47:999-1005, 1987; Wood et al.,Nature 314:446-449, 1985; and Shaw et al., J. Natl Cancer Inst.,80:1553-1559, 1988).

Methods for humanizing antibodies have been described in the art. Inpractice, humanized antibodies are typically human antibodies in whichsome hypervariable region residues and possibly some framework regionresidues are substituted by residues from analogous sites in rodentantibodies. Accordingly, such “humanized” antibodies are chimericantibodies wherein substantially less than an intact human variableregion has been substituted by the corresponding sequence from anonhuman species. To a degree, this can be accomplished in connectionwith techniques of humanization and display techniques using appropriatelibraries. It will be appreciated that murine antibodies or antibodiesfrom other species can be humanized or primatized using techniques wellknown in the art (see e.g., Winter et al., Immunol Today, 14:43-46,1993; and Wright et al., Crit. Reviews in Immunol., 12125-168, 1992).The antibody of interest may be engineered by recombinant DNA techniquesto substitute the CH1, CH2, CH3, hinge domains, and/or the frameworkdomain with the corresponding human sequence (see WO 92/02190 and U.S.Pat. Nos. 5,530,101, 5,585,089, 5,693,761, 5,693,792, 5,714,350, and5,777,085). Also, the use of Ig cDNA for construction of chimericimmunoglobulin genes is known in the art (Liu et al., P.N.A.S. 84:3439,1987; J. Immunol. 139:3521, 1987). mRNA is isolated from a hybridoma orother cell producing the antibody and used to produce cDNA. The cDNA ofinterest may be amplified by the polymerase chain reaction usingspecific primers (U.S. Pat. Nos. 4,683,195 and 4,683,202).Alternatively, a library is made and screened to isolate the sequence ofinterest. The DNA sequence encoding the variable region of the antibodyis then fused to human constant region sequences. The sequences of humanconstant regions to genes may be found in Kabat et al. (1991) Sequencesof Proteins of Immunological Interest, N.I.H. publication no. 91-3242.Human C region genes are readily available from known clones. The choiceof isotype will be guided by the desired effector functions, such ascomplement fixation, or activity in antibody-dependent cellularcytotoxicity. In various embodiments, the isotype is selected from thegroup consisiting of IgG1, IgG2, IgG3 and IgG4. Either of the humanlight chain constant regions, kappa or lambda, may be used. Thechimeric, humanized antibody is then expressed by conventional methods.

U.S. Pat. No. 5,693,761 to Queen et al, discloses a refinement on Winteret al. for humanizing antibodies, and is based on the premise thatascribes avidity loss to problems in the structural motifs in thehumanized framework which, because of steric or other chemicalincompatibility, interfere with the folding of the CDRs into thebinding-capable conformation found in the mouse antibody. To addressthis problem, Queen teaches using human framework sequences closelyhomologous in linear peptide sequence to framework sequences of themouse antibody to be humanized. Accordingly, the methods of Queen focuson comparing framework sequences between species. Typically, allavailable human variable region sequences are compared to a particularmouse sequence and the percentage identity between correspondentframework residues is calculated. The human variable region with thehighest percentage is selected to provide the framework sequences forthe humanizing project. Queen also teaches that it is important toretain in the humanized framework, certain amino acid residues from themouse framework critical for supporting the CDRs in a binding-capableconformation. Potential criticality is assessed from molecular models.Candidate residues for retention are typically those adjacent in linearsequence to a CDR or physically within 6 Å of any CDR residue.

In other approaches, the importance of particular framework amino acidresidues is determined experimentally once a low-avidity humanizedconstruct is obtained, by reversion of single residues to the mousesequence and assaying antigen-binding as described by Riechmann et al,1988. Another example approach for identifying important amino acids inframework sequences is disclosed by U.S. Pat. No. 5,821,337 to Carter etal, and by U.S. Pat. No. 5,859,205 to Adair et al. These referencesdisclose specific Kabat residue positions in the framework, which, in ahumanized antibody may require substitution with the correspondent mouseamino acid to preserve avidity.

Another method of humanizing antibodies, referred to as “frameworkshuffling”, relies on generating a combinatorial library with nonhumanCDR variable regions fused in frame into a pool of individual humangermline frameworks (Dall'Acqua et al., Methods, 36:43, 2005). Thelibraries are then screened to identify clones that encode humanizedantibodies which retain good binding.

The choice of human variable regions, both light and heavy, to be usedin making the desired humanized antibodies is very important to reduceantigenicity. According to the so-called “best-fit” method, the sequenceof the variable region of a rodent antibody is screened against theentire library of known human variable-domain sequences. The humansequence that is closest to that of the rodent is then accepted as thehuman framework region (framework region) for the humanized antibody(Sims et al., J. Immunol., 151:2296, 1993; Chothia et al., J. Mol.Biol., 196:901, 1987). Another method uses a particular framework regionderived from the consensus sequence of all human antibodies of aparticular subgroup of light or heavy chain variable regions. The sameframework may be used for several different humanized antibodies (Carteret al., Proc. Natl. Acad. Sci. USA, 89:4285, 1992; Presta et al., J.Immunol., 151:2623, 1993).

The choice of nonhuman residues to substitute into the human variableregion can be influenced by a variety of factors. These factors include,for example, the rarity of the amino acid in a particular position, theprobability of interaction with either the CDRs or the antigen, and theprobability of participating in the interface between the light andheavy chain variable domain interface. (See, for example, U.S. Pat. Nos.5,693,761, 6,632,927, and 6,639,055). One method to analyze thesefactors is through the use of three-dimensional models of the nonhumanand humanized sequences. Three-dimensional immunoglobulin models arecommonly available and are familiar to those skilled in the art.Computer programs are available that illustrate and display probablethree-dimensional conformational structures of selected candidateimmunoglobulin sequences. Inspection of these displays permits analysisof the likely role of the residues in the functioning of the candidateimmunoglobulin sequence, e.g., the analysis of residues that influencethe ability of the candidate immunoglobulin to bind its antigen. In thisway, nonhuman residues can be selected and substituted for humanvariable region residues in order to achieve the desired antibodycharacteristic, such as increased affinity for the target antigen(s).

Methods for making fully human antibodies have been described in theart. By way of example, a method for producing an anti-CGRP antibody orantigen-binding fragment thereof comprises the steps of synthesizing alibrary of human antibodies on phage, screening the library with CGRP oran antibody-binding portion thereof, isolating phage that bind CGRP, andobtaining the antibody from the phage. By way of another example, onemethod for preparing the library of antibodies for use in phage displaytechniques comprises the steps of immunizing a non-human animalcomprising human immunoglobulin loci with CGRP or an antigenic portionthereof to create an immune response, extracting antibody-producingcells from the immunized animal; isolating RNA encoding heavy and lightchains of antibodies of the invention from the extracted cells, reversetranscribing the RNA to produce cDNA, amplifying the cDNA using primers,and inserting the cDNA into a phage display vector such that antibodiesare expressed on the phage. Recombinant anti-CGRP antibodies of theinvention may be obtained in this way.

Recombinant human anti-CGRP antibodies of the invention can also beisolated by screening a recombinant combinatorial antibody library.Preferably the library is a scFv phage display library, generated usinghuman VL and VH cDNAs prepared from mRNA isolated from B cells. Methodsfor preparing and screening such libraries are known in the art. Kitsfor generating phage display libraries are commercially available (e.g.,the Pharmacia Recombinant Phage Antibody System, catalog no. 27-9400-01;and the Stratagene SurfZAP™ phage display kit, catalog no. 240612).There also are other methods and reagents that can be used in generatingand screening antibody display libraries (see, e.g., U.S. Pat. No.5,223,409; PCT Publication Nos. WO 92/18619, WO 91/17271, WO 92/20791,WO 92/15679, WO 93/01288, WO 92/01047, WO 92/09690; Fuchs et al.,Bio/Technology 9:1370-1372 (1991); Hay et al., Hum. Antibod. Hybridomas3:81-85, 1992; Huse et al., Science 246:1275-1281, 1989; McCafferty etal., Nature 348:552-554, 1990; Griffiths et al., EMBO J. 12:725-734,1993; Hawkins et al., J. Mol. Biol. 226:889-896, 1992; Clackson et al.,Nature 352:624-628, 1991; Gram et al., Proc. Natl. Acad. Sci. USA89:3576-3580, 1992; Garrad et al., Bio/Technology 9:1373-1377, 1991;Hoogenboom et al., Nuc. Acid Res. 19:4133-4137, 1991; and Barbas et al.,Proc. Natl. Acad. Sci. USA 88:7978-7982, 1991, each incorporated hereinby reference for purposes of teaching preparation and screening of phasedisplay libraries.

Human antibodies are also produced by immunizing a non-human, transgenicanimal comprising within its genome some or all of human immunoglobulinheavy chain and light chain loci with a human IgE antigen, e.g., aXenoMouse™ animal (Abgenix, Inc./Amgen, Inc.—Fremont, Calif.).XenoMouse™ mice are engineered mouse strains that comprise largefragments of human immunoglobulin heavy chain and light chain loci andare deficient in mouse antibody production. See, e.g., Green et al.,Nature Genetics 7:13-21, 1994; and U.S. Pat. Nos. 5,916,771, 5,939,598,5,985,615, 5,998,209, 6,075,181, 6,091,001, 6,114,598, 6,130,364,6,162,963 and 6,150,584. See also WO 91/10741, WO 94/02602, WO 96/34096,WO 96/33735, WO 98/16654, WO 98/24893, WO 98/50433, WO 99/45031, WO99/53049, WO 00/09560, and WO 00/037504. XenoMouse™ mice produce anadult-like human repertoire of fully human antibodies and generateantigen-specific human antibodies. In some embodiments, the XenoMouse™mice contain approximately 80% of the human antibody V gene repertoirethrough introduction of megabase sized, germline configuration fragmentsof the human heavy chain loci and kappa light chain loci in yeastartificial chromosome (YAC). In other embodiments, XenoMouse™ micefurther contain approximately all of the human lambda light chain locus.See Mendez et al., Nature Genetics 15:146-156, 1997, Green andJakobovits, J. Exp. Med. 188:483-495 (1998), and WO 98/24893 (eachincorporated by reference in its entirety for purposes of teaching thepreparation of fully human antibodies). In another aspect, the presentinvention provides a method for making anti-CGRP antibodies fromnon-human, non-mouse animals by immunizing non-human transgenic animalsthat comprise human immunoglobulin loci with a CGRP antigen. One canproduce such animals using the methods described in the above-citeddocuments.

Characterization of Antibody Binding to Antigen

Antibodies of the present invention can be tested for binding to humanCGRP by, for example, standard ELISA. As an example, microtiter platesare coated with purified CGRP in PBS, and then blocked with 5% bovineserum albumin in PBS. Dilutions of antibody (e.g., dilutions of plasmafrom CGRP-immunized mice) are added to each well and incubated for 1-2hours at 37° C. The plates are washed with PBS/Tween and then incubatedwith secondary reagent (e.g., for human antibodies, a goat-anti-humanIgG Fc-specific polyclonal reagent) conjugated to alkaline phosphatasefor 1 hour at 37° C. After washing, the plates are developed with pNPPsubstrate (1 mg/ml), and analyzed at OD of 405-650. Preferably, micewhich develop the highest titers will be used for fusions. An ELISAassay can also be used to screen for hybridomas that show positivereactivity with CGRP immunogen. Hybridomas that bind with high avidityto CGRP are subcloned and further characterized. One clone from eachhybridoma, which retains the reactivity of the parent cells (by ELISA),can be chosen for making a 5-10 vial cell bank stored at −140° C., andfor antibody purification.

To determine if the selected anti-CGRP monoclonal antibodies bind tounique epitopes, each antibody can be biotinylated using commerciallyavailable reagents (Pierce, Rockford, Ill.). Competition studies usingunlabeled monoclonal antibodies and biotinylated monoclonal antibodiescan be performed using CGRP coated-ELISA plates as described above.Biotinylated mAb binding can be detected with a strep-avidin-alkalinephosphatase probe. To determine the isotype of purified antibodies,isotype ELISAs can be performed using reagents specific for antibodiesof a particular isotype. For example, to determine the isotype of ahuman monoclonal antibody, wells of microtiter plates can be coated with1.mu.g/ml of anti-human immunoglobulin overnight at 4.degree. C. Afterblocking with 1% BSA, the plates are reacted with 1 μg/ml or less oftest monoclonal antibodies or purified isotype controls, at ambienttemperature for one to two hours. The wells can then be reacted witheither human IgG1 or human IgM-specific alkaline phosphatase-conjugatedprobes. Plates are developed and analyzed as described above.

Anti-CGRP human IgGs can be further tested for reactivity with CGRPantigen by Western blotting. Briefly, CGRP can be prepared and subjectedto sodium dodecyl sulfate polyacrylamide gel electrophoresis. Afterelectrophoresis, the separated antigens are transferred tonitrocellulose membranes, blocked with 10% fetal calf serum, and probedwith the monoclonal antibodies to be tested. Human IgG binding can bedetected using anti-human IgG alkaline phosphatase and developed withBCIP/NBT substrate tablets (Sigma Chem. Co., St. Louis, Mo.).

Identification of Anti-CGRP Antibodies

The present invention provides monoclonal antibodies, andantigen-binding fragments thereof, that specifically bind to CGRPantigen.

Further included in the present invention are antibodies that bind tothe same epitope as the anti-CGRP antibodies of the present invention.To determine if an antibody can compete for binding to the same epitopeas the epitope bound by the anti-CGRP antibodies of the presentinvention, a cross-blocking assay, e.g., a competitive ELISA assay, canbe performed. In an exemplary competitive ELISA assay, CGRP coated onthe wells of a microtiter plate is pre-incubated with or withoutcandidate competing antibody and then the biotin-labeled anti-CGRPantibody of the invention is added. The amount of labeled anti-CGRPantibody bound to the CGRP antigen in the wells is measured usingavidin-peroxidase conjugate and appropriate substrate. The antibody canbe labeled with a radioactive or fluorescent label or some otherdetectable and measurable label. The amount of labeled anti-CGRPantibody that bound to the antigen will have an indirect correlation tothe ability of the candidate competing antibody (test antibody) tocompete for binding to the same epitope, i.e., the greater the affinityof the test antibody for the same epitope, the less labeled antibodywill be bound to the antigen-coated wells. A candidate competingantibody is considered an antibody that binds substantially to the sameepitope or that competes for binding to the same epitope as an anti-CGRPantibody of the invention if the candidate antibody can block binding ofthe CGRP antibody by at least 20%, preferably by at least 20-50%, evenmore preferably, by at least 50% as compared to the control performed inparallel in the absence of the candidate competing antibody. It will beunderstood that variations of this assay can be performed to arrive atthe same quantitative value.

The amino acid sequences of the heavy chain CDRs and the light chainCDRs of three murine antibodies, 6G5C8C4 (also referred to hereinafteras “A1”), 6G5C8C4 (also referred to hereinafter as “A2”), and 6G5C8C4(also referred to hereinafter as “A3”), generated as described herein,are shown below in Table 2.

TABLE 2 Heavy Chain CDRs Ab HCDR1 HCDR2 HCDR3 A1 DYYMN DINPNNGITIVPYYFDY (SEQ ID NO: 2) GTTYNQKFKG (SEQ ID NO: 6) (SEQ ID NO: 3) A2DYYMN DVNPNNG ITIVPLYFDF (SEQ ID NO: 2) DTTYNQKFKG (SEQ ID NO: 7)(SEQ ID NO: 4) A3 DYYMN DVNPNNG ITIVPVYFDY (SEQ ID NO: 2) GTHYNQKFKG(SEQ ID NO: 8) (SEQ ID NO: 5) Light Chain CDRs Ab LCDR1 LCDR2 LCDR3 A1KASQNVGSNVA SASFRYS QQYNSYPYT (SEQ ID NO: 9) (SEQ ID NO: 12)(SEQ ID NO: 14) A2 KASQNVGINVA SASYRYS QQYNTYPYT (SEQ ID NO: 10)(SEQ ID NO: 13) (SEQ ID NO: 15) A3 KASQNVGPNVA SASYRYS QQYNYYPYT(SEQ ID NO: 11) (SEQ ID NO: 13) (SEQ ID NO: 16)

In various embodiments of the present invention, the antibody orantigen-binding fragment is a murine antibody, 6G5C8C4 (“A1”),comprising the heavy chain variable region sequence of SEQ ID NO: 17:

(SEQ ID NO: 17) EVQLQQSGPELVKPGASVKISCKASGYTFTDYYMNWVKQSHGKSLEWIGDINPNNGGTTYNQKFKGKATLTVDKSSTTAYMELRSLTSEDSAVYYCAVITIVPYYFDYWGQGTTLTVSSand the light chain variable region sequence of SEQ ID NO: 23:

(SEQ ID NO: 23) DIVMTQSQKFMSTSVGDRVSVTCKASQNVGSNVAWYQQKPGQSPKALIYSASFRYSRVPDRFSGSGSGTDFTLTISNVQSED LADYFCQQYNSYPYTFGAGTKLELK

In certain alternative embodiments, the antibody is an antibodycomprising a heavy chain and a light chain, wherein heavy chaincomprises a heavy chain variable region, and wherein the heavy chainvariable region comprises a sequence that has at least about 80%, atleast about 85%, at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, or at least about 99% identity to the amino acid sequence as setforth in SEQ ID NO: 17, or its corresponding polynucleotide sequence SEQID NO: 18:

(SEQ ID NO: 18) gaggtccagctgcaacaatctggacctgagctggtgaagcctggggcttcagtgaagatatcctgtaaggcttctggatacacgttcactgactactacatgaactgggtgaagcagagccatggaaagagccttgagtggattggagatattaatcctaacaatggtggtactacttacaaccagaagttcaagggcaaggccacattgactgtagacaagtcctccaccacagcctacatggagctccgcagcctgacatctgaggactctgcagtctattactgtgcagtcattactatagtcccctactactttgactactggggccaaggc accactctcacagtctcctcaand wherein the light chain comprises a light chain variable region, andwherein the light chain variable region comprises a sequence that has atleast about 75%, at least about 80%, at least about 85%, at least about90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least about 99%identity to the amino acid sequence as set forth in SEQ ID NO: 23, orits corresponding polynucleotide sequence SEQ ID NO: 24:

(SEQ ID NO: 24) gacattgttatgacccagtctcaaaaattcatgtccacatcagtaggagacagggtcagcgtcacctgcaaggccagtcagaatgtgggtagtaatgtagcctggtatcaacagaaaccagggcaatctcctaaagcactgatttactcggcatcctttcgttacagtagagtccctgatcgcttctcaggcagtggatctgggacagatttcactctcaccatcagcaatgtgcagtctgaagacttggcagactatttctg tcagcaatataacagttatccttacacgtt cggtgctgggaccaagctggagctgaaa

In various embodiments of the present invention, the antibody orantigen-binding fragment is a murine antibody, 24G7B10G2 (“A2”),comprising the heavy chain variable region sequence of SEQ ID NO: 19:

(SEQ ID NO: 19) EVQLLQSGPELMKPGTSVKISCKASGYTFTDYYMNWVKQSHGKSLEWIGDVNPNNGDTTYNQKFKGKATLTIDKSSSTAYMELRSLTSEDSAVYYCASII IVPLYFDFWGQGTTLAVSS and the light chain variable region sequence of SEQ ID NO: 25:

(SEQ ID NO: 25) DIVMTQSQKFMSTSVGDRVSVTCKASQNVGINVAWYQQKPGQSPKALLYSASYRYSGVPDRFTGSGSGTDFTLTISNVKSGDLAEYFCQQYNTYPYTFGG GTKLEIK 

In certain alternative embodiments, the antibody is an antibodycomprising a heavy chain and a light chain, wherein heavy chaincomprises a heavy chain variable region, and wherein the heavy chainvariable region comprises a sequence that has at least about 80%, atleast about 85%, at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, or at least about 99% identity to the amino acid sequence as setforth in SEQ ID NO: 19, or its corresponding polynucleotide sequence SEQID NO: 20:

(SEQ ID NO: 20) gaggtccagctgttacaatctggacctgagctgatgaagcctgggacttcagtgaagatatcctgtaaggcttctggatacacgttcactgactactacatgaactgggtgaagcagagccatggtaagagccttgagtggattggagatgttaatcctaacaatggtgatactacctacaaccagaagttcaagggcaaggccacattgactatagacaagtcctccagcacagcctacatggaactccgcagcctgacatctgaggactctgcagtctactactgtgcaagtattacgattgttcctctttactttgacttctccgcccaacccaccactctcccact ctcctca and wherein the light chain comprises a light chain variable region, andwherein the light chain variable region comprises a sequence that has atleast about 75%, at least about 80%, at least about 85%, at least about90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least about 99%identity to the amino acid sequence as set forth in SEQ ID NO: 25, orits corresponding polynucleotide sequence SEQ ID NO: 26:

(SEQ ID NO: 26) gacattgtgatgacccagtctcaaaaattcatgtccacatcagtgggagacagggtcagcgtcacctgcaaggccagtcagaatgtgggtattaatgtagcctggtatcaacagaagccaggacaatctcctaaagcactgctttactcggcatcctaccgatatagtggagtccctgatcgcttcacaggcagtggttctgggacagatttcactctcaccatcagcaatgtgaagtctggagacttggcagagtatttctgtcagcaatataacacctatccgtacacgttcggaggggggaccaagctggaaataaaa 

In various embodiments of the present invention, the antibody orantigen-binding fragment is a murine antibody, 30B8G9B11 (“A3”),comprising the heavy chain variable region sequence of SEQ ID NO: 21:

(SEQ ID NO: 21) EVQLQQSGPELVKPGASVKISCKASGYTFTDYYMNWVKQSHGKSLEWIGDVNPNNGGTHYNQKFKGKATLTVDKSSSTAYMELRSLTSEDSAVYYCASIT IVPVYFDFWGQGTTLTVSS and the light chain variable region sequence of SEQ ID NO: 27:

(SEQ ID NO: 27) DIVMTQSQKFMSTSVGDRVSVTCKASQNVGPNVAWYRQKPGQSPKALIYSASYRYSGVPDRFTGSGSGTDFTLTISNVQSEDLADYICQQYNYYPYTFGG GTKLEIK 

In certain alternative embodiments, the antibody is an antibodycomprising a heavy chain and a light chain, wherein heavy chaincomprises a heavy chain variable region, and wherein the heavy chainvariable region comprises a sequence that has at least about 80%, atleast about 85%, at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, or at least about 99% identity to the amino acid sequence as setforth in SEQ ID NO: 21, or its corresponding polynucleotide sequence SEQID NO: 22:

(SEQ ID NO: 22) gaggtccagctgcaacaatctggacctgaactggtgaagcctggggcttcagtgaagatatcctgtaaggcttctggatacacgttcactgactactacatgaactgggtgaagcagagccatggaaagagccttgagtggattggagatgttaatcctaacaatggtggtactcactacaaccagaagttcaagggcaaggccacattgactgtagacaagtcctccagtacagcctacatggagctccgcagcctgacatctgaggactctgcagtctattactgtgcaagtattacgattgtacctgtttactttgacttctggggccaaggcaccactctcacagt ctcctca and wherein the light chain comprises a light chain variable region, andwherein the light chain variable region comprises a sequence that has atleast about 75%, at least about 80%, at least about 85%, at least about90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least about 99%identity to the amino acid sequence as set forth in SEQ ID NO: 27, orits corresponding polynucleotide sequence SEQ ID NO: 28:

(SEQ ID NO: 28) gacattgtgatgacccagtctcaaaaattcatgtccacatcagtaggagacagggtcagcgtcacctgcaaggccagtcagaatgtgggtcctaatgtagcctggtatcgacagaaaccaggacaatctcctaaagctctgatttactcggcatcctaccggtacagtggagtccctgatcgcttcacaggcagtggatctgggacagatttcactctcaccatcagcaatgtgcaatctgaagacttggcagactatatctgtcagcagtataactactatccgtacacgttcggaggggggaccaaactggaaataaaa 

In various embodiments, the heavy chain variable domain comprises asequence of amino acids that is encoded by a polynucleotide thathybridizes under moderately stringent conditions to the complement of apolynucleotide that encodes a heavy chain variable domain having thesequence of SEQ ID NOs: 18, 20 or 22. In another embodiment, the heavychain variable domain comprises a sequence of amino acids that isencoded by a polynucleotide that hybridizes under stringent conditionsto the complement of a polynucleotide that encodes a heavy chainvariable domain having the sequence of SEQ ID NOs: 18, 20 or 22. Inanother embodiment, the light chain variable domain comprises a sequenceof amino acids that is encoded by a polynucleotide that hybridizes undermoderately stringent conditions to the complement of a polynucleotidethat encodes a light chain variable domain having the sequence of SEQ IDNOs: 24, 26 or 28. In another embodiment, the light chain variabledomain comprises a sequence of amino acids that is encoded by apolynucleotide that hybridizes under stringent conditions to thecomplement of a polynucleotide that encodes a light chain variabledomain having the sequence of SEQ ID NOs: 24, 26 or 28.

In various embodiments, antibodies of the present invention includeantibodies that bind to the same epitope as murine antibody A1. Invarious embodiments, antibodies of the present invention includeantibodies that bind to the same epitope as murine antibody A2. Invarious embodiments, antibodies of the present invention includeantibodies that bind to the same epitope as murine antibody A3.

In various embodiments of the present invention, the antibody orantigen-binding fragment is a murine-human chimeric antibody derivedfrom murine antibody A3 and human IgG4 comprising the heavy chainsequence of SEQ ID NO: 29 and wherein amino acids 1-19 are a leadersequence:

(SEQ ID NO: 29) MGWSWILLFLLSVTAGVHSEVQLQQSGPELVKPGASVKISCKASGYTFTDYYMNWVKQSHGKSLEWIGDVNPNNGGTHYNQKFKGKATLTVDKSSSTAYMELRSLTSEDSAVYYCASITIVPVYFDFWGQGTTLTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTIPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEAL HNHYTQKSLSLSLGK and the light chain sequence of SEQ ID NO: 31 and wherein amino acids1-19 are a leader sequence:

(SEQ ID NO: 31) MGWSWILLFLLSVTAGVHSDIVMTQSQKFMSTSVGDRVSVTCKASQNVGPNVAWYRQKPGQSPKALIYSASYRYSGVPDRFTGSGSGTDFTLTISNVQSEDLADYICQQYNYYPYTFGGGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC 

In certain alternative embodiments, the antibody is a murine-humanchimeric antibody comprising a heavy chain and a light chain, whereinthe heavy chain comprises a sequence that has at least about 80%, atleast about 85%, at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, or at least about 99% identity to the amino acid sequence as setforth in SEQ ID NO: 29, or its corresponding polynucleotide sequence SEQID NO: 30:

(SEQ ID NO: 30) atgggctggagctggatcctgctgttcctcctgagcgtgacagcaggagtgcacagcgaggtccagctgcaacaatctggacctgaactggtgaagcctggggcttcagtgaagatatcctgtaaggcttctggatacacgttcactgactactacatgaactgggtgaagcagagccatggaaagagccttgagtggattggagatgttaatcctaacaatggtggtactcactacaaccagaagttcaagggcaaggccacattgactgtagacaagtcctccagtacagcctacatggagctccgcagcctgacatctgaggactctgcagtctattactgtgcaagtattacgattgtacctgtttactttgacttctggggccaaggcaccactctcacagtctcctcagcctctacaaagggcccctccgtgtttccactggctccctgcagcaggtctacatccgagagcaccgctgctctgggatgtctggtgaaggattacttccctgagccagtgaccgtgagctggaactccggagctctgacatccggagtgcacacctttcctgctgtgctgcagagctctggcctgtacagcctgtccagcgtggtgacagtgccatcttccagcctgggcaccaagacatatacctgcaacgtggaccataagcccagcaataccaaggtggataagagagtggagtctaagtacggaccaccttgcccaccatgtccagctcctgagtttctgggaggaccatccgtgttcctgtttcctccaaagcctaaggacaccctgatgatctctcgcacacccgaggtgacctgtgtggtggtggacgtgtcccaggaggatcctgaggtgcagttcaactggtacgtggatggcgtggaggtgcacaatgctaagaccaagcctagggaggagcagtttaacagcacataccgggtggtgtctgtgctgaccgtgctgcatcaggactggctgaacggcaaggagtataagtgcaaggtgagcaataagggcctgccatcttccatcgagaagacaatctctaaggctaagggacagcctagggagccacaggtgtacaccctgcccccttcccaggaggagatgacaaagaaccaggtgagcctgacctgtctggtgaagggcttctatccttctgacatcgctgtggagtgggagtccaatggccagccagagaacaattacaagaccacaccacccgtgctggactccgatggcagcttctttctgtattccaggctgaccgtggataagagccggtggcaggagggcaatgtgttttcttgttccgtgatgcacgaagcactgcacaaccactacactcagaagtccctgtcactgtccctgggcaag and wherein the light chain comprises a sequence that has at least about75%, at least about 80%, at least about 85%, at least about 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least about 99% identity to theamino acid sequence as set forth in SEQ ID NO: 31, or its correspondingpolynucleotide sequence SEQ ID NO: 32:

(SEQ ID NO: 32) atgggctggagctggatcctgctgttcctcctgagcgtgacagcaggagtgcacagcgaggtccagctgttacaatctggacctgagctgatgaagcctgggacttcagtgaagatatcctgtaaggcttctggatacacgttcactgactactacatgaactgggtgaagcagagccatggtaagagccttgagtggattggagatgttaatcctaacaatggtgatactacctacaaccagaagttcaagggcaaggccacattgactatagacaagtcctccagcacagcctacatggaactccgcagcctgacatctgaggactctgcagtctactactgtgcaagtattacgattgttcctctttactttgacttctggggccaaggcaccactctcgcagtctcctcacgaacggtggctgcaccatctgtcttcatcttcccgccatctgatgagcagttgaaatctggaactgcctctgttgtgtgcctgctgaataacttctatcccagagaggccaaagtacagtggaaggtggataacgccctccaatcgggtaactcccaggagagtgtcacagagcaggacagcaaggacagcacctacagcctcagcagcaccctgacgctgagcaaagcagactacgagaaacacaaagtctacgcctgcgaagtcacccatcagggcctgagctcgcccgtcacaaagagcttcaacaggggagagtgt 

In various embodiments, an isolated humanized antibody orantigen-binding fragment thereof of the present invention binds to humanCGRP and comprises a heavy chain variable region having a sequenceidentical, substantially identical or substantially similar to SEQ IDNOs: 33, 35, 37 and 39, and a light chain variable region having thesequence identical, substantially identical or substantially similar toSEQ ID NOs: 34, 36, 38 and 40. In various embodiments the antibody is ahumanized antibody or antigen-binding fragment thereof which comprisesthe heavy chain variable region having the amino acid sequence set forthin SEQ ID NO: 33 and the light chain variable region having the aminoacid sequence set forth in SEQ ID NO: 34. In various embodiments theantibody is a humanized antibody or antigen-binding fragment thereofwhich comprises the heavy chain variable region having the amino acidsequence set forth in SEQ ID NO: 33 and the light chain variable regionhaving the amino acid sequence set forth in SEQ ID NO: 36. In variousembodiments the antibody is a humanized antibody or antigen-bindingfragment thereof which comprises the heavy chain variable region havingthe amino acid sequence set forth in SEQ ID NO: 33 and the light chainvariable region having the amino acid sequence set forth in SEQ ID NO:38. In various embodiments the antibody is a humanized antibody orantigen-binding fragment thereof which comprises the heavy chainvariable region having the amino acid sequence set forth in SEQ ID NO:33 and the light chain variable region having the amino acid sequenceset forth in SEQ ID NO: 40. In various embodiments the antibody is ahumanized antibody or antigen-binding fragment thereof which comprisesthe heavy chain variable region having the amino acid sequence set forthin SEQ ID NO: 35 and the light chain variable region having the aminoacid sequence set forth in SEQ ID NO: 34. In various embodiments theantibody is a humanized antibody or antigen-binding fragment thereofwhich comprises the heavy chain variable region having the amino acidsequence set forth in SEQ ID NO: 35 and the light chain variable regionhaving the amino acid sequence set forth in SEQ ID NO: 36. In variousembodiments the antibody is a humanized antibody or antigen-bindingfragment thereof which comprises the heavy chain variable region havingthe amino acid sequence set forth in SEQ ID NO: 35 and the light chainvariable region having the amino acid sequence set forth in SEQ ID NO:38. In various embodiments the antibody is a humanized antibody orantigen-binding fragment thereof which comprises the heavy chainvariable region having the amino acid sequence set forth in SEQ ID NO:35 and the light chain variable region having the amino acid sequenceset forth in SEQ ID NO: 40. In various embodiments the antibody is ahumanized antibody or antigen-binding fragment thereof which comprisesthe heavy chain variable region having the amino acid sequence set forthin SEQ ID NO: 37 and the light chain variable region having the aminoacid sequence set forth in SEQ ID NO: 34. In various embodiments theantibody is a humanized antibody or antigen-binding fragment thereofwhich comprises the heavy chain variable region having the amino acidsequence set forth in SEQ ID NO: 37 and the light chain variable regionhaving the amino acid sequence set forth in SEQ ID NO: 36. In variousembodiments the antibody is a humanized antibody or antigen-bindingfragment thereof which comprises the heavy chain variable region havingthe amino acid sequence set forth in SEQ ID NO: 37 and the light chainvariable region having the amino acid sequence set forth in SEQ ID NO:38. In various embodiments the antibody is a humanized antibody orantigen-binding fragment thereof which comprises the heavy chainvariable region having the amino acid sequence set forth in SEQ ID NO:37 and the light chain variable region having the amino acid sequenceset forth in SEQ ID NO: 40. In various embodiments the antibody is ahumanized antibody or antigen-binding fragment thereof which comprisesthe heavy chain variable region having the amino acid sequence set forthin SEQ ID NO: 39 and the light chain variable region having the aminoacid sequence set forth in SEQ ID NO: 34. In various embodiments theantibody is a humanized antibody or antigen-binding fragment thereofwhich comprises the heavy chain variable region having the amino acidsequence set forth in SEQ ID NO: 39 and the light chain variable regionhaving the amino acid sequence set forth in SEQ ID NO: 36. In variousembodiments the antibody is a humanized antibody or antigen-bindingfragment thereof which comprises the heavy chain variable region havingthe amino acid sequence set forth in SEQ ID NO: 39 and the light chainvariable region having the amino acid sequence set forth in SEQ ID NO:38. In various embodiments the antibody is a humanized antibody orantigen-binding fragment thereof which comprises the heavy chainvariable region having the amino acid sequence set forth in SEQ ID NO:39 and the light chain variable region having the amino acid sequenceset forth in SEQ ID NO: 40.

In various embodiments, the antibodies or antigen-binding fragmentsthereof comprise a heavy chain variable domain comprising a sequence ofamino acids that differs from the sequence of a heavy chain variabledomain having the amino acid sequence set forth in SEQ ID NOs: 33, 35,37 and 39 only at 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1 or 0residues, wherein each such sequence difference is independently eithera deletion, insertion, or substitution of one amino acid residue. Invarious embodiments, the antibodies or antigen-binding fragments thereofcomprise a heavy chain variable domain comprising a sequence that has atleast about 80%, at least about 85%, at least about 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, or at least about 99% identity to the aminoacid sequence as set forth in SEQ ID NOs: 33, 35, 37 and 39.

In various embodiments, an isolated humanized antibody orantigen-binding fragment thereof of the present invention binds to humanCGRP and comprises sequence of amino acids that differs from thesequence of a light chain variable domain having the amino acid sequenceset forth in SEQ ID NOs: 34, 36, 38 and 40 only at 15, 14, 13, 12, 11,10, 9, 8, 7, 6, 5, 4, 3, 2, 1 or 0 residues, wherein each such sequencedifference is independently either a deletion, insertion, orsubstitution of one amino acid residue. In various embodiments, theantibodies or antigen-binding fragments thereof comprise a light chainvariable domain comprising a sequence that has at least about 80%, atleast about 85%, at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, or at least about 99% identity to the amino acid sequence as setforth in SEQ ID NOs: 34, 36, 38 and 40.

In various embodiments of the present invention, the antibody is ahumanized IgG comprising the heavy chain sequence (“H1”) of SEQ ID NO:41 and wherein amino acids 1-19 are a leader sequence:

(SEQ ID NO: 41) MGWSWILLFLLSVTAGVHSQVQLVQSGAEVKKPGASVKVSCKASGYTFTDYYMNWVRQAPGQGLEWMGDVNPNNGGTHYNQKFKGRVTMTRDTSISTAYMELSRLRSDDTAVYYCARITIVPVYFDFWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTIPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEAL HNHYTQKSLSLSLGK and the light chain sequence (“L1”) of SEQ ID NO: 49 and wherein aminoacids 1-19 are a leader sequence:

(SEQ ID NO: 49) MGWSWILLFLLSVTAGVHSDIQLTQSPSFLSASVGDRVTITCKASQNVGPNVAWYQQKPGKAPKLLIYSASYRYSGVPSRFSGSGSGTEFTLTISSLQPEDFATYYCQQYNYYPYTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC 

In certain alternative embodiments, the antibody is an antibodycomprising a heavy chain and a light chain, wherein the heavy chaincomprises a sequence that has at least about 80%, at least about 85%, atleast about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or at leastabout 99% identity to the amino acid sequence as set forth in SEQ ID NO:41, or its corresponding polynucleotide sequence SEQ ID NO: 42:

(SEQ ID NO: 42) atgggctggagctggatcctgctgttcctcctgagcgtgacagcaggagtgcacagccaggtccagctggtccagtcaggggcagaagtgaagaaacccggagcaagtgtgaaggtgtcatgcaaagcctcaggatatacattcacagactactatatgaactgggtgcgacaggcaccaggacagggactggagtggatgggggatgtcaaccctaacaatggcgggactcactacaatcagaagttcaaaggcagggtgaccatgacacgcgacactagcatctccaccgcctatatggaactgtctcggctgagaagcgacgataccgccgtctactattgcgctagaattactattgtgcccgtgtattttgatttttggggacagggaactctggtcaccgtctcatccgcctctacaaagggcccctccgtgtttccactggctccctgcagcaggtctacatccgagagcaccgctgctctgggatgtctggtgaaggattacttccctgagccagtgaccgtgagctggaactccggagctctgacatccggagtgcacacctttcctgctgtgctgcagagctctggcctgtacagcctgtccagcgtggtgacagtgccatcttccagcctgggcaccaagacatatacctgcaacgtggaccataagcccagcaataccaaggtggataagagagtggagtctaagtacggaccaccttgcccaccatgtccagctcctgagtttctgggaggaccatccgtgttcctgtttcctccaaagcctaaggacaccctgatgatctctcgcacacccgaggtgacctgtgtggtggtggacgtgtcccaggaggatcctgaggtgcagttcaactggtacgtggatggcgtggaggtgcacaatgctaagaccaagcctagggaggagcagtttaacagcacataccgggtggtgtctgtgctgaccgtgctgcatcaggactggctgaacggcaaggagtataagtgcaaggtgagcaataagggcctgccatcttccatcgagaagacaatctctaaggctaagggacagcctagggagccacaggtgtacaccctgcccccttcccaggaggagatgacaaagaaccaggtgagcctgacctgtctggtgaagggcttctatccttctgacatcgctgtggagtgggagtccaatggccagccagagaacaattacaagaccacaccacccgtgctggactccgatggcagcttctttctgtattccaggctgaccgtggataagagccggtggcaggagggcaatgtgttttcttgttccgtgatgcacgaagcactgcacaaccactacactcagaagtccctgtcactgtccctgggcaag and wherein the light chain comprises a sequence that has at least about75%, at least about 80%, at least about 85%, at least about 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least about 99% identity to theamino acid sequence as set forth in SEQ ID NO: 49, or its correspondingpolynucleotide sequence SEQ ID NO: 50:

(SEQ ID NO: 50) atgggctggagctggatcctgctgttcctcctgagcgtgacagcaggagtgcacagcgacatccagctgacccagtccccctcattcctgtccgcaagtgtgggcgaccgagtcaccatcacctgtaaggcaagccagaacgtgggccccaacgtggcatggtaccagcagaagcccgggaaagcccctaagctgctgatctattctgctagttaccggtattctggcgtcccaagcagattctcaggcagcgggtccggaactgagtttaccctgacaattagctccctgcagcccgaagacttcgccacctactattgtcagcagtacaactattacccatacaccttcgggcagggcactaaactggaaatcaaacgaacggtggctgcaccatctgtcttcatcttcccgccatctgatgagcagttgaaatctggaactgcctctgttgtgtgcctgctgaataacttctatcccagagaggccaaagtacagtggaaggtggataacgccctccaatcgggtaactcccaggagagtgtcacagagcaggacagcaaggacagcacctacagcctcagcagcaccctgacgctgagcaaagcagactacgagaaacacaaagtctacgcctgcgaagtcacccatcagggcctgagctcgcccgtcacaaagagcttcaacaggggagagtgt

In various embodiments of the present invention, the antibody is ahumanized IgG comprising the heavy chain sequence (“H2”) of SEQ ID NO:43 and wherein amino acids 1-19 are a leader sequence:

(SEQ ID NO: 43) MGWSWILLFLLSVTAGVHSQVQLVQSGAEVVKPGASVKVSCKASGYTFTDYYMNWVRQAPGQGLEWMGDVNPNNGGTHYNQKFKGRVTMTVDTSISTAYMELSRLRSDDTAVYYCASITIVPVYFDFWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTIPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEAL HNHYTQKSLSLSLGKand the light chain sequence (“L2”) of SEQ ID NO: 51 and wherein aminoacids 1-19 are a leader sequence:

(SEQ ID NO: 51) MGWSWILLFLLSVTAGVHSDIQLTQSPSFLSASVGDRVTITCKASQNVGPNVAWYRQKPGKAPKALIYSASYRYSGVPSRFSGSGSGTEFTLTISSLOPEDFATYICQQYNYYPYTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

In certain alternative embodiments, the antibody is an antibodycomprising a heavy chain and a light chain, wherein the heavy chaincomprises a sequence that has at least about 80%, at least about 85%, atleast about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or at leastabout 99% identity to the amino acid sequence as set forth in SEQ ID NO:43, or its corresponding polynucleotide sequence SEQ ID NO: 44:

(SEQ ID NO: 44) atgggctggagctggatcctgctgttcctcctgagcgtgacagcaggagtgcacagccaggtccagctggtccagtcaggagcagaggtcgtgaaacccggagcaagcgtcaaggtctcatgcaaagcaagcggctatacattcacagactactatatgaactgggtgaggcaggcaccaggacagggactggagtggatgggggatgtcaaccctaacaatggcgggactcactacaatcagaagttcaaaggccgggtgaccatgacagtcgacactagcatctccaccgcctatatggaactgtctcggctgagaagtgacgataccgccgtgtactattgcgcttccattactatcgtgcccgtctactttgacttctggggacaggggacactggtgaccgtctcatccgcctctacaaagggcccctccgtgtttccactggctccctgcagcaggtctacatccgagagcaccgctgctctgggatgtctggtgaaggattacttccctgagccagtgaccgtgagctggaactccggagctctgacatccggagtgcacacctttcctgctgtgctgcagagctctggcctgtacagcctgtccagcgtggtgacagtgccatcttccagcctgggcaccaagacatatacctgcaacgtggaccataagcccagcaataccaaggtggataagagagtggagtctaagtacggaccaccttgcccaccatgtccagctcctgagtttctgggaggaccatccgtgttcctgtttcctccaaagcctaaggacaccctgatgatctctcgcacacccgaggtgacctgtgtggtggtggacgtgtcccaggaggatcctgaggtgcagttcaactggtacgtggatggcgtggaggtgcacaatgctaagaccaagcctagggaggagcagtttaacagcacataccgggtggtgtctgtgctgaccgtgctgcatcaggactggctgaacggcaaggagtataagtgcaaggtgagcaataagggcctgccatcttccatcgagaagacaatctctaaggctaagggacagcctagggagccacaggtgtacaccctgcccccttcccaggaggagatgacaaagaaccaggtgagcctgacctgtctggtgaagggcttctatccttctgacatcgctgtggagtgggagtccaatggccagccagagaacaattacaagaccacaccacccgtgctggactccgatggcagcttctttctgtattccaggctgaccgtggataagagccggtggcaggagggcaatgtgttttcttgttccgtgatgcacgaagcactgcacaaccactacactcagaagtccctgtcactgtccctgggcaagand wherein the light chain comprises a sequence that has at least about75%, at least about 80%, at least about 85%, at least about 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least about 99% identity to theamino acid sequence as set forth in SEQ ID NO: 51, or its correspondingpolynucleotide sequence SEQ ID NO: 52:

(SEQ ID NO: 52) atgggctggagctggatcctgctgttcctcctgagcgtgacagcaggagtgcacagcgacatccagctgacccagtccccatccttcctgagcgcaagcgtcggagacagagtgaccattacctgcaaagcatcccagaacgtgggccccaacgtggcatggtacaggcagaagcccgggaaagcccctaaggctctgatctattctgccagttaccggtattctggcgtcccaagcagattctcaggcagcgggtccggaactgagtttaccctgacaatcagctccctgcagcccgaagacttcgctacctacatttgtcagcagtacaactattatccttacacctttgggcagggcactaaactggaaatcaagcgaacggtggctgcaccatctgtcttcatcttcccgccatctgatgagcagttgaaatctggaactgcctctgttgtgtgcctgctgaataacttctatcccagagaggccaaagtacagtggaaggtggataacgccctccaatcgggtaactcccaggagagtgtcacagagcaggacagcaaggacagcacctacagcctcagcagcaccctgacgctgagcaaagcagactacgagaaacacaaagtctacgcctgcgaagtcacccatcagggcctgagctcgcccgtcacaaagagcttcaacaggggagagtgt

In various embodiments of the present invention, the antibody is ahumanized IgG comprising the heavy chain sequence (“H3”) of SEQ ID NO:45 and wherein amino acids 1-19 are a leader sequence:

(SEQ ID NO: 45) MGWSWILLFLLSVTAGVHSQVQLVQSGAEVVKPGASVKVSCKASGYTFTDYYMNWVKQAPGQGLEWIGDVNPNNGGTHYNQKFKGRVTLTVDTSISTAYMELSRLRSDDTAVYYCASITIVPVYFDFWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTIPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEAL HNHYTQKSLSLSLGKand the light chain sequence (“L3”) of SEQ ID NO: 53 and wherein aminoacids 1-19 are a leader sequence:

(SEQ ID NO: 53) MGWSWILLFLLSVTAGVHSDIQLTQSPSFLSTSVGDRVTITCKASQNVGPNVAWYRQKPGKSPKALIYSASYRYSGVPSRFSGSGSGTEFTLTISSLQPEDFATYICQQYNYYPYTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

In certain alternative embodiments, the antibody is an antibodycomprising a heavy chain and a light chain, wherein the heavy chaincomprises a sequence that has at least about 80%, at least about 85%, atleast about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or at leastabout 99% identity to the amino acid sequence as set forth in SEQ ID NO:45, or its corresponding polynucleotide sequence SEQ ID NO: 46:

(SEQ ID NO: 46) atgggctggagctggatcctgctgttcctcctgagcgtgacagcaggagtgcacagccaggtccagctggtccagtcaggggcagaggtggtcaaacccggagcaagcgtgaaagtctcatgcaaagcaagcggctatacattcacagactactatatgaactgggtgaagcaggcaccaggacagggactggagtggatcggggatgtcaaccctaacaatggcgggactcactacaatcagaagttcaaaggcagggtgaccctgacagtcgacactagcatctccaccgcctatatggaactgtctcggctgagaagtgacgataccgccgtgtactattgcgcttccattactattgtgcccgtctattttgacttctggggacaggggacactggtgaccgtctcctcagcctctacaaagggcccctccgtgtttccactggctccctgcagcaggtctacatccgagagcaccgctgctctgggatgtctggtgaaggattacttccctgagccagtgaccgtgagctggaactccggagctctgacatccggagtgcacacctttcctgctgtgctgcagagctctggcctgtacagcctgtccagcgtggtgacagtgccatcttccagcctgggcaccaagacatatacctgcaacgtggaccataagcccagcaataccaaggtggataagagagtggagtctaagtacggaccaccttgcccaccatgtccagctcctgagtttctgggaggaccatccgtgttcctgtttcctccaaagcctaaggacaccctgatgatctctcgcacacccgaggtgacctgtgtggtggtggacgtgtcccaggaggatcctgaggtgcagttcaactggtacgtggatggcgtggaggtgcacaatgctaagaccaagcctagggaggagcagtttaacagcacataccgggtggtgtctgtgctgaccgtgctgcatcaggactggctgaacggcaaggagtataagtgcaaggtgagcaataagggcctgccatcttccatcgagaagacaatctctaaggctaagggacagcctagggagccacaggtgtacaccctgcccccttcccaggaggagatgacaaagaaccaggtgagcctgacctgtctggtgaagggcttctatccttctgacatcgctgtggagtgggagtccaatggccagccagagaacaattacaagaccacaccacccgtgctggactccgatggcagcttctttctgtattccaggctgaccgtggataagagccggtggcaggagggcaatgtgttttcttgttccgtgatgcacgaagcactgcacaaccactacactcagaagtccctgtcactgtccctgggcaag and wherein the light chain comprises a sequence that has at least about75%, at least about 80%, at least about 85%, at least about 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least about 99% identity to theamino acid sequence as set forth in SEQ ID NO: 53, or its correspondingpolynucleotide sequence SEQ ID NO: 54:

(SEQ ID NO: 54) atgggctggagctggatcctgctgttcctcctgagcgtgacagcaggagtgcacagcgacatccagctgacccagagcccttccttcctgagcacaagcgtcggagatagagtcacaatcacctgtaaagcaagccagaacgtgggccccaacgtggcatggtacaggcagaagcccgggaaaagccctaaggccctgatctattctgctagttaccggtattctggcgtcccaagcagattctcaggcagcgggtccggaactgagtttaccctgacaatcagctccctgcagcccgaagacttcgccacctacatttgtcagcagtacaactattacccatacaccttcgggcaggggaccaaactggaaatcaagcgaacggtggctgcaccatctgtcttcatcttcccgccatctgatgagcagttgaaatctggaactgcctctgttgtgtgcctgctgaataacttctatcccagagaggccaaagtacagtggaaggtggataacgccctccaatcgggtaactcccaggagagtgtcacagagcaggacagcaaggacagcacctacagcctcagcagcaccctgacgctgagcaaagcagactacgagaaacacaaagtctacgcctgcgaagtcacccatcagggcctgagctcgcccgtcacaaagagcttcaacaggggagagtgt

In various embodiments of the present invention, the antibody is ahumanized IgG comprising the heavy chain sequence (“H4”) of SEQ ID NO:47 and wherein amino acids 1-19 are a leader sequence:

(SEQ ID NO: 47) MGWSWILLFLLSVTAGVHSQVQLVQSGAEVVKPGASVKISCKASGYTFTDYYMNWVKQAPGQGLEWIGDVNPNNGGTHYNQKFKGRATLTVDTSISTAYMELSRLRSDDTAVYYCASITIVPVYFDFWGQGTLLTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTIPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEAL HNHYTQKSLSLSLGKand the light chain sequence (“L4”) of SEQ ID NO: 55 and wherein aminoacids 1-19 are a leader sequence:

(SEQ ID NO: 55) MGWSWILLFLLSVTAGVHSDIQMTQSPSFLSTSVGDRVIVICKASQNVGPNVAWYRQKPGKSPKALIYSASYRYSGVPSRFSGSGSGTEFTLTISSVQPEDFATYICQQYNYYPYTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

In certain alternative embodiments, the antibody is an antibodycomprising a heavy chain and a light chain, wherein the heavy chaincomprises a sequence that has at least about 80%, at least about 85%, atleast about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or at leastabout 99% identity to the amino acid sequence as set forth in SEQ ID NO:47, or its corresponding polynucleotide sequence SEQ ID NO: 48:

(SEQ ID NO: 48) atgggctggagctggatcctgctgttcctcctgagcgtgacagcaggagtgcacagccaggtgcagctggtccagtcaggggcagaggtcgtcaaacccggagcaagcgtgaagattagttgtaaggcatcaggctatactttcacagactactatatgaactgggtgaagcaggctccaggacagggactggagtggatcggggatgtcaaccctaacaatggcgggactcactacaatcagaagttcaaaggcagggcaaccctgacagtggacactagcatctccaccgcctatatggaactgtctcggctgagaagtgacgataccgccgtctactattgcgcttccattaccattgtgccagtctattttgatttttggggacagggaactctgctgacagtctcctcagcctctacaaagggcccctccgtgtttccactggctccctgcagcaggtctacatccgagagcaccgctgctctgggatgtctggtgaaggattacttccctgagccagtgaccgtgagctggaactccggagctctgacatccggagtgcacacctttcctgctgtgctgcagagctctggcctgtacagcctgtccagcgtggtgacagtgccatcttccagcctgggcaccaagacatatacctgcaacgtggaccataagcccagcaataccaaggtggataagagagtggagtctaagtacggaccaccttgcccaccatgtccagctcctgagtttctgggaggaccatccgtgttcctgtttcctccaaagcctaaggacaccctgatgatctctcgcacacccgaggtgacctgtgtggtggtggacgtgtcccaggaggatcctgaggtgcagttcaactggtacgtggatggcgtggaggtgcacaatgctaagaccaagcctagggaggagcagtttaacagcacataccgggtggtgtctgtgctgaccgtgctgcatcaggactggctgaacggcaaggagtataagtgcaaggtgagcaataagggcctgccatcttccatcgagaagacaatctctaaggctaagggacagcctagggagccacaggtgtacaccctgcccccttcccaggaggagatgacaaagaaccaggtgagcctgacctgtctggtgaagggcttctatccttctgacatcgctgtggagtgggagtccaatggccagccagagaacaattacaagaccacaccacccgtgctggactccgatggcagcttctttctgtattccaggctgaccgtggataagagccggtggcaggagggcaatgtgttttcttgttccgtgatgcacgaagcactgcacaaccactacactcagaagtccctgtcactgtccctgggcaagand wherein the light chain comprises a sequence that has at least about75%, at least about 80%, at least about 85%, at least about 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least about 99% identity to theamino acid sequence as set forth in SEQ ID NO: 55, or its correspondingpolynucleotide sequence SEQ ID NO: 56:

(SEQ ID NO: 56) atgggctggagctggatcctgctgttcctcctgagcgtgacagcaggagtgcacagcgacatccagatgacccagtccccctccttcctgagcacaagcgtgggcgatagagtcaccgtcacctgtaaagcaagccagaacgtgggccccaacgtggcatggtacaggcagaagcccgggaaaagccctaaggccctgatctattctgctagttaccggtattctggcgtgccaagcagattctcaggcagcgggtccggaactgagtttaccctgacaatcagctccgtccagcccgaagacttcgccacctacatttgtcagcagtacaactattacccatacaccttcgggcaggggactaaactggaaatcaagcgaacggtggctgcaccatctgtcttcatcttcccgccatctgatgagcagttgaaatctggaactgcctctgttgtgtgcctgctgaataacttctatcccagagaggccaaagtacagtggaaggtggataacgccctccaatcgggtaactcccaggagagtgtcacagagcaggacagcaaggacagcacctacagcctcagcagcaccctgacgctgagcaaagcagactacgagaaacacaaagtctacgcctgcgaagtcacccatcagggcctgagctcgcccgtcacaaagagcttcaacaggggagagtgt

In various embodiments of the present disclosure, the antibody may be ananti-CGRP antibody that has the same or higher antigen-binding affinityas that of the antibody comprising the heavy chain sequence as set forthin any of SEQ ID NOs: 41, 43, 45 and 47. In various embodiments, theantibody may be an anti-CGRP antibody which binds to the same epitope asthe antibody comprising the heavy chain sequence as set forth in any ofSEQ ID NOs: 41, 43, 45 and 47. In various embodiments, the antibody isan anti-CGRP antibody which competes with the antibody comprising theheavy chain sequence as set forth in any of SEQ ID NOs: 41, 43, 45 and47. In various embodiments, the antibody may be an anti-CGRP antibodywhich comprises at least one (such as two or three) CDRs of the heavychain sequence as set forth in any of SEQ ID NOs: 41, 43, 45 and 47.

In various embodiments, the antibody contains an heavy chain amino acidsequence that shares an observed homology of, e.g., at least 70%, atleast 75%, at least 80%, at least 85%, at least 90%, at least 95%, atleast 96%, at least 97%, at least 98%, or at least 99% with any of SEQID NOs: 41, 43, 45 and 47. In various embodiments, the antibody containsa heavy chain nucleic acid sequence that shares an observed homology of,e.g., at least 70%, at least 75%, at least 80%, at least 85%, at least90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least99% with any of SEQ ID NOs: 42, 44, 46 and 48.

In various embodiments of the present disclosure the antibody may be ananti-CGRP antibody that has the same or higher antigen-binding affinityas that of the antibody comprising the light chain sequence as set forthin any of SEQ ID NOs: 49, 51, 53 and 55. In various embodiments, theantibody may be an anti-CGRP antibody which binds to the same epitope asthe antibody comprising the light chain sequence as set forth in any ofSEQ ID NOs: 49, 51, 53 and 55. In various embodiments, the antibody isan anti-CGRP antibody which competes with the antibody comprising thelight chain sequence as set forth in any of SEQ ID NOs: 49, 51, 53 and55. In various embodiments, the antibody may be an anti-CGRP antibodywhich comprises at least one (such as two or three) CDRs of the lightchain sequence as set forth in any of SEQ ID NOs: 49, 51, 53 and 55.

In various embodiments, the antibody contains an light chain amino acidsequence that shares an observed homology of, e.g., at least 70%, atleast 75%, at least 80%, at least 85%, at least 90%, at least 95%, atleast 96%, at least 97%, at least 98%, or at least 99% with any of SEQID NOs: 49, 51, 53 and 55. In various embodiments, the antibody containsa nucleic acid sequence that shares an observed homology of, e.g., atleast 70%, at least 75%, at least 80%, at least 85%, at least 90%, atleast 95%, at least 96%, at least 97%, at least 98%, or at least 99%with any of SEQ ID NOs: 50, 52, 54 and 56.

In various embodiments, an isolated humanized antibody orantigen-binding fragment thereof of the present invention binds to humanCGRP and comprises a heavy chain variable region having a sequenceidentical, substantially identical or substantially similar to SEQ IDNOs: 33, 35, 37 and 39, and a light chain variable region having thesequence identical, substantially identical or substantially similar toSEQ ID NOs: 34, 36, 38 and 40. In various embodiments, an isolatedhumanized antibody or antigen-binding fragment thereof of the presentinvention binds to human CGRP and comprises the heavy chain sequence setforth in SEQ ID NO: 41, and the light chain sequence set forth in SEQ IDNO: 49. In various embodiments, an isolated humanized antibody orantigen-binding fragment thereof of the present invention binds to humanCGRP and comprises the heavy chain sequence set forth in SEQ ID NO: 41,and the light chain sequence set forth in SEQ ID NO: 51. In variousembodiments, an isolated humanized antibody or antigen-binding fragmentthereof of the present invention binds to human CGRP and comprises theheavy chain sequence set forth in SEQ ID NO: 41, and the light chainsequence set forth in SEQ ID NO: 53. In various embodiments, an isolatedhumanized antibody or antigen-binding fragment thereof of the presentinvention binds to human CGRP and comprises the heavy chain sequence setforth in SEQ ID NO: 41, and the light chain sequence set forth in SEQ IDNO: 55. In various embodiments, an isolated humanized antibody orantigen-binding fragment thereof of the present invention binds to humanCGRP and comprises the heavy chain sequence set forth in SEQ ID NO: 43,and the light chain sequence set forth in SEQ ID NO: 49. In variousembodiments, an isolated humanized antibody or antigen-binding fragmentthereof of the present invention binds to human CGRP and comprises theheavy chain sequence set forth in SEQ ID NO: 43, and the light chainsequence set forth in SEQ ID NO: 51. In various embodiments, an isolatedhumanized antibody or antigen-binding fragment thereof of the presentinvention binds to human CGRP and comprises the heavy chain sequence setforth in SEQ ID NO: 43, and the light chain sequence set forth in SEQ IDNO: 53. In various embodiments, an isolated humanized antibody orantigen-binding fragment thereof of the present invention binds to humanCGRP and comprises the heavy chain sequence set forth in SEQ ID NO: 43,and the light chain sequence set forth in SEQ ID NO: 55. In variousembodiments, an isolated humanized antibody or antigen-binding fragmentthereof of the present invention binds to human CGRP and comprises theheavy chain sequence set forth in SEQ ID NO: 45, and the light chainsequence set forth in SEQ ID NO: 49. In various embodiments, an isolatedhumanized antibody or antigen-binding fragment thereof of the presentinvention binds to human CGRP and comprises the heavy chain sequence setforth in SEQ ID NO: 45, and the light chain sequence set forth in SEQ IDNO: 51. In various embodiments, an isolated humanized antibody orantigen-binding fragment thereof of the present invention binds to humanCGRP and comprises the heavy chain sequence set forth in SEQ ID NO: 45,and the light chain sequence set forth in SEQ ID NO: 53. In variousembodiments, an isolated humanized antibody or antigen-binding fragmentthereof of the present invention binds to human CGRP and comprises theheavy chain sequence set forth in SEQ ID NO: 45, and the light chainsequence set forth in SEQ ID NO: 55. In various embodiments, an isolatedhumanized antibody or antigen-binding fragment thereof of the presentinvention binds to human CGRP and comprises the heavy chain sequence setforth in SEQ ID NO: 47, and the light chain sequence set forth in SEQ IDNO: 49. In various embodiments, an isolated humanized antibody orantigen-binding fragment thereof of the present invention binds to humanCGRP and comprises the heavy chain sequence set forth in SEQ ID NO: 47,and the light chain sequence set forth in SEQ ID NO: 51. In variousembodiments, an isolated humanized antibody or antigen-binding fragmentthereof of the present invention binds to human CGRP and comprises theheavy chain sequence set forth in SEQ ID NO: 47, and the light chainsequence set forth in SEQ ID NO: 53. In various embodiments, an isolatedhumanized antibody or antigen-binding fragment thereof of the presentinvention binds to human CGRP and comprises the heavy chain sequence setforth in SEQ ID NO: 47, and the light chain sequence set forth in SEQ IDNO: 55.

Antibodies or antigen-binding fragments thereof of the invention cancomprise any constant region known in the art. The light chain constantregion can be, for example, a kappa- or lambda-type light chain constantregion, e.g., a human kappa- or lambda-type light chain constant region.The heavy chain constant region can be, for example, an alpha-, delta-,epsilon-, gamma-, or mu-type heavy chain constant regions, e.g., a IgA-,IgD-, IgE-, IgG- and IgM-type heavy chain constant region. In variousembodiments, the light or heavy chain constant region is a fragment,derivative, variant, or mutein of a naturally occurring constant region.

Techniques are known for deriving an antibody of a different subclass orisotype from an antibody of interest, i.e., subclass switching. Thus,IgG antibodies may be derived from an IgM antibody, for example, andvice versa. Such techniques allow the preparation of new antibodies thatpossess the antigen-binding properties of a given antibody (the parentantibody), but also exhibit biological properties associated with anantibody isotype or subclass different from that of the parent antibody.Recombinant DNA techniques may be employed. Cloned DNA encodingparticular antibody polypeptides may be employed in such procedures,e.g., DNA encoding the constant domain of an antibody of the desiredisotype. See also Lanitto et al., Methods Mol. Biol. 178:303-16, 2002.

In various embodiments, an antibody of the invention further comprises alight chain kappa or lambda constant domain, or a fragment thereof, andfurther comprises a heavy chain constant domain, or a fragment thereof.Sequences of the light chain constant region and heavy chain constantregion used in the exemplified antibodies, and polynucleotides encodingthem, are provided below.

Light Chain (Kappa) Constant Region (SEQ ID NO: 57)RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTK SFNRGECLight Chain (Lambda) Constant Region (SEQ ID NO: 58)GQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADSSPVKAGVETTTPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTV APTECSHeavy Chain Constant Region (SEQ ID NO: 59)ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK

Antibodies of the present invention may also be described or specifiedin terms of their cross-reactivity. Antibodies that bind CGRPs, whichhave at least 95%, at least 90%, at least 85%, at least 80%, at least75%, at least 70%, at least 65%, at least 60%, at least 55%, and atleast 50% identity (as calculated using methods known in the art anddescribed herein) to human CGRP are also included in the presentinvention.

Further included in the present invention are antibodies that bind tothe same epitope as the anti-CGRP antibodies of the present invention.To determine if an antibody can compete for binding to the same epitopeas the epitope bound by the anti-CGRP antibodies of the presentinvention, a cross-blocking assay, e.g., a competitive ELISA assay, canbe performed. In an exemplary competitive ELISA assay, CGRP coated onthe wells of a microtiter plate is pre-incubated with or withoutcandidate competing antibody and then the biotin-labeled anti-CGRPantibody of the invention is added. The amount of labeled anti-CGRPantibody bound to the CGRP antigen in the wells is measured usingavidin-peroxidase conjugate and appropriate substrate. The antibody canbe labeled with a radioactive or fluorescent label or some otherdetectable and measurable label. The amount of labeled anti-CGRPantibody that bound to the antigen will have an indirect correlation tothe ability of the candidate competing antibody (test antibody) tocompete for binding to the same epitope, i.e., the greater the affinityof the test antibody for the same epitope, the less labeled antibodywill be bound to the antigen-coated wells. A candidate competingantibody is considered an antibody that binds substantially to the sameepitope or that competes for binding to the same epitope as an anti-CGRPantibody of the invention if the candidate antibody can block binding ofthe CGRP antibody by at least 20%, by at least 30%, by at least 40%, orby at least 50% as compared to the control performed in parallel in theabsence of the candidate competing antibody. It will be understood thatvariations of this assay can be performed to arrive at the samequantitative value.

In certain alternative embodiments, the antibodies of the presentinvention can be engineered by modifying one or more residues within oneor both variable regions (i.e., VH and/or VL), or by modifying residueswithin the constant region(s), e.g., to alter the effector function(s)of the antibody. In various embodiments, the variable region of theantibody will by modified by performing CDR grafting using frameworksequences can be obtained from public DNA databases or publishedreferences that include germline antibody gene sequences (e.g.,Tomlinson, I. M., et al., J. Mol. Biol. 227:776-798, 1992; and Cox, J.P. L. et al., Eur. J. Immunol. 24:827-836, 1994; the contents of each ofwhich are expressly incorporated herein by reference). In variousembodiments, the antibodies may be modified using site-directedmutagenesis or PCR-mediated mutagenesis to introduce a mutation(s) inthe VH and/or VL which improves binding affinity and/or decreasesimmunogenicity. In various embodiments, the antibodies may be modifiedin the Fc region for purposes of altering the serum half-life,complement fixation, Fc receptor binding, and/or antigen-dependentcellular cytotoxicity of the antibody. In various embodiments, theantibodies may be modified for purposes of modifying the glycosylationof the antibody. Methods for performing each of the modificationsdescribed herein, and others, are well known to the skilled artisan.

Pharmaceutical Compositions

In another aspect, the present invention provides a pharmaceuticalcomposition comprising an antibody or antigen-binding fragment thereofas described above. The pharmaceutical compositions, methods and uses ofthe invention thus also encompass embodiments of combinations(co-administration) with other active agents, as detailed below.

Generally, the antibodies, or antigen-binding fragments thereofantibodies of the present invention are suitable to be administered as aformulation in association with one or more pharmaceutically acceptableexcipient(s). The term ‘excipient’ is used herein to describe anyingredient other than the compound(s) of the invention. The choice ofexcipient(s) will to a large extent depend on factors such as theparticular mode of administration, the effect of the excipient onsolubility and stability, and the nature of the dosage form. As usedherein, “pharmaceutically acceptable excipient” includes any and allsolvents, dispersion media, coatings, antibacterial and antifungalagents, isotonic and absorption delaying agents, and the like that arephysiologically compatible. Some examples of pharmaceutically acceptableexcipients are water, saline, phosphate buffered saline, dextrose,glycerol, ethanol and the like, as well as combinations thereof. In manycases, it will be preferable to include isotonic agents, for example,sugars, polyalcohols such as mannitol, sorbitol, or sodium chloride inthe composition. Additional examples of pharmaceutically acceptablesubstances are wetting agents or minor amounts of auxiliary substancessuch as wetting or emulsifying agents, preservatives or buffers, whichenhance the shelf life or effectiveness of the antibody. Pharmaceuticalcompositions of the present invention and methods for their preparationwill be readily apparent to those skilled in the art. Such compositionsand methods for their preparation may be found, for example, inRemington's Pharmaceutical Sciences, 19th Edition (Mack PublishingCompany, 1995). Pharmaceutical compositions are preferably manufacturedunder GMP conditions.

A pharmaceutical composition of the invention may be prepared, packaged,or sold in bulk, as a single unit dose, or as a plurality of single unitdoses. As used herein, a “unit dose” is discrete amount of thepharmaceutical composition comprising a predetermined amount of theactive ingredient. The amount of the active ingredient is generallyequal to the dosage of the active ingredient which would be administeredto a subject or a convenient fraction of such a dosage such as, forexample, one-half or one-third of such a dosage.

Any method for administering peptides, proteins or antibodies acceptedin the art may suitably be employed for the antibodies and portions ofthe invention.

The pharmaceutical compositions of the invention are typically suitablefor parenteral administration. As used herein, “parenteraladministration” of a pharmaceutical composition includes any route ofadministration characterized by physical breaching of a tissue of asubject and administration of the pharmaceutical composition through thebreach in the tissue, thus generally resulting in the directadministration into the blood stream, into muscle, or into an internalorgan. Parenteral administration thus includes, but is not limited to,administration of a pharmaceutical composition by injection of thecomposition, by application of the composition through a surgicalincision, by application of the composition through a tissue-penetratingnon-surgical wound, and the like. In particular, parenteraladministration is contemplated to include, but is not limited to,subcutaneous, intraperitoneal, intramuscular, intrasternal, intravenous,intraarterial, intrathecal, intraventricular, intraurethral,intracranial, intrasynovial injection or infusions; and kidney dialyticinfusion techniques. Various embodiments include the intravenous and thesubcutaneous routes.

Formulations of a pharmaceutical composition suitable for parenteraladministration typically generally comprise the active ingredientcombined with a pharmaceutically acceptable carrier, such as sterilewater or sterile isotonic saline. Such formulations may be prepared,packaged, or sold in a form suitable for bolus administration or forcontinuous administration. Injectable formulations may be prepared,packaged, or sold in unit dosage form, such as in ampoules or inmulti-dose containers containing a preservative. Formulations forparenteral administration include, but are not limited to, suspensions,solutions, emulsions in oily or aqueous vehicles, pastes, and the like.Such formulations may further comprise one or more additionalingredients including, but not limited to, suspending, stabilizing, ordispersing agents. In one embodiment of a formulation for parenteraladministration, the active ingredient is provided in dry (i.e. powder orgranular) form for reconstitution with a suitable vehicle (e.g. sterilepyrogen-free water) prior to parenteral administration of thereconstituted composition. Parenteral formulations also include aqueoussolutions which may contain excipients such as salts, carbohydrates andbuffering agents (preferably to a pH of from 3 to 9), but, for someapplications, they may be more suitably formulated as a sterilenon-aqueous solution or as a dried form to be used in conjunction with asuitable vehicle such as sterile, pyrogen-free water. Exemplaryparenteral administration forms include solutions or suspensions insterile aqueous solutions, for example, aqueous propylene glycol ordextrose solutions. Such dosage forms can be suitably buffered, ifdesired. Other parentally-administrable formulations which are usefulinclude those which comprise the active ingredient in microcrystallineform, or in a liposomal preparation. Formulations for parenteraladministration may be formulated to be immediate and/or modifiedrelease. Modified release formulations include delayed-, sustained-,pulsed-, controlled-, targeted and programmed release.

For example, in one aspect, sterile injectable solutions can be preparedby incorporating the anti-CGRP antibody in the required amount in anappropriate solvent with one or a combination of ingredients enumeratedabove, as required, followed by filtered sterilization. Generally,dispersions are prepared by incorporating the active compound into asterile vehicle that contains a basic dispersion medium and the requiredother ingredients from those enumerated above. In the case of sterilepowders for the preparation of sterile injectable solutions, thepreferred methods of preparation are vacuum drying and freeze-dryingthat yields a powder of the active ingredient plus any additionaldesired ingredient from a previously sterile-filtered solution thereof.The proper fluidity of a solution can be maintained, for example, by theuse of a coating such as lecithin, by the maintenance of the requiredparticle size in the case of dispersion and by the use of surfactants.Prolonged absorption of injectable compositions can be brought about byincluding in the composition an agent that delays absorption, forexample, monostearate salts and gelatin.

The antibodies of the invention can also be administered intranasally orby inhalation, typically in the form of a dry powder (either alone, as amixture, or as a mixed component particle, for example, mixed with asuitable pharmaceutically acceptable excipient) from a dry powderinhaler, as an aerosol spray from a pressurized container, pump, spray,atomizer (preferably an atomizer using electrohydrodynamics to produce afine mist), or nebulizer, with or without the use of a suitablepropellant, or as nasal drops.

The pressurized container, pump, spray, atomizer, or nebulizer generallycontains a solution or suspension of an antibody of the inventioncomprising, for example, a suitable agent for dispersing, solubilizing,or extending release of the active, a propellant(s) as solvent.

Prior to use in a dry powder or suspension formulation, the drug productis generally micronized to a size suitable for delivery by inhalation(typically less than 5 microns). This may be achieved by any appropriatecomminuting method, such as spiral jet milling, fluid bed jet milling,supercritical fluid processing to form nanoparticles, high pressurehomogenization, or spray drying.

Capsules, blisters and cartridges for use in an inhaler or insufflatormay be formulated to contain a powder mix of the compound of theinvention, a suitable powder base and a performance modifier.

Suitable flavours, such as menthol and levomenthol, or sweeteners, suchas saccharin or saccharin sodium, may be added to those formulations ofthe invention intended for inhaled/intranasal administration.

Formulations for inhaled/intranasal administration may be formulated tobe immediate- and/or modified release. Modified release formulationsinclude delayed-, sustained-, pulsed-, controlled-, targeted andprogrammed release.

In the case of dry powder inhalers and aerosols, the dosage unit isdetermined by means of a valve which delivers a metered amount. Units inaccordance with the invention are typically arranged to administer ametered dose or “puff” of an antibody of the invention. The overalldaily dose will typically be administered in a single dose or, moreusually, as divided doses throughout the day.

The antibodies and antibody portions of the invention may also beformulated for an oral route administration. Oral administration mayinvolve swallowing, so that the compound enters the gastrointestinaltract, and/or buccal, lingual, or sublingual administration by which thecompound enters the blood stream directly from the mouth.

Formulations suitable for oral administration include solid, semi-solidand liquid systems such as tablets; soft or hard capsules containingmulti- or nano-particulates, liquids, or powders; lozenges (includingliquid-filled); chews; gels; fast dispersing dosage forms; films;ovules; sprays; and buccal/mucoadhesive patches.

Pharmaceutical compositions intended for oral use may be preparedaccording to any method known to the art for the manufacture ofpharmaceutical compositions and such compositions may contain one ormore agents selected from the group consisting of sweetening agents inorder to provide a pharmaceutically elegant and palatable preparation.For example, to prepare orally deliverable tablets, the antibody orantigen-binding fragment thereof is mixed with at least onepharmaceutical excipient, and the solid formulation is compressed toform a tablet according to known methods, for delivery to thegastrointestinal tract. The tablet composition is typically formulatedwith additives, e.g. a saccharide or cellulose carrier, a binder such asstarch paste or methyl cellulose, a filler, a disintegrator, or otheradditives typically usually used in the manufacture of medicalpreparations. To prepare orally deliverable capsules, DHEA is mixed withat least one pharmaceutical excipient, and the solid formulation isplaced in a capsular container suitable for delivery to thegastrointestinal tract. Compositions comprising antibodies orantigen-binding fragments thereof may be prepared as described generallyin Remington's Pharmaceutical Sciences, 18th Ed. 1990 (Mack PublishingCo. Easton Pa. 18042) at Chapter 89, which is herein incorporated byreference.

In various embodiments, the pharmaceutical compositions are formulatedas orally deliverable tablets containing antibodies or antigen-bindingfragments thereof in admixture with non-toxic pharmaceuticallyacceptable excipients which are suitable for manufacture of tablets.These excipients may be inert diluents, such as calcium carbonate,sodium carbonate, lactose, calcium phosphate or sodium phosphate;granulating and disintegrating agents, for example, maize starch,gelatin or acacia, and lubricating agents, for example, magnesiumstearate, stearic acid, or talc. The tablets may be uncoated or they maybe coated with known techniques to delay disintegration and absorptionin the gastrointestinal track and thereby provide a sustained actionover a longer period of time. For example, a time delay material such asglyceryl monostearate or glyceryl distearate alone or with a wax may beemployed.

In various embodiments, the pharmaceutical compositions are formulatedas hard gelatin capsules wherein the antibody or antigen-bindingfragment thereof is mixed with an inert solid diluent, for example,calcium carbonate, calcium phosphate, or kaolin or as soft gelatincapsules wherein the antibody or antigen-binding fragment thereof ismixed with an aqueous or an oil medium, for example, arachis oil, peanutoil, liquid paraffin or olive oil.

Liquid formulations include suspensions, solutions, syrups and elixirs.Such formulations may be employed as fillers in soft or hard capsules(made, for example, from gelatin or hydroxypropylmethylcellulose) andtypically comprise a carrier, for example, water, ethanol, polyethyleneglycol, propylene glycol, methylcellulose, or a suitable oil, and one ormore emulsifying agents and/or suspending agents. Liquid formulationsmay also be prepared by the reconstitution of a solid, for example, froma sachet.

Therapeutic Uses

In another aspect, the present invention relates to methods of treatinga subject suffering from a CGRP-associated disorder, comprisingadministering to said subject a therapeutically effective amount of anantibody or antigen-binding fragment thereof of the present invention.In various embodiments, the subject is a human subject. In variousembodiments, the CGRP-associated disorder is selected from the groupconsisting of headaches, hot flushes, chronic pain, type II diabetesmellitus, functional bowel disorders or an inflammatory bowel diseases,diarrhea, psoriasis, pain and itch associated with arthritis and skindisease, cardiovascular disorders, and hemodynamic derangementassociated with endotoxemia, sepsis, obesity, diabetes and arthritis.

In various embodiments, the a CGRP-associated disorder is a headacheselected from the group consisting of: migraine with or without aura;hemiplegic migraine; cluster headaches; migrainous neuralgia; chronicheadaches; tension headaches; headaches resulting from other medicalconditions (such as infection or increased pressure in the skull due toa tumor); chronic paroxysmal hemicrania; miscellaneous headacheunassociated with a structural lesion; headache associated with anon-vascular intracranial disorder; headache associated with theadministration of a substance or its withdrawal; headache associatedwith noncephalic infection; headache associated with a metabolicdisorder; headache associated with a disorder of the cranium, neck,eyes, ears, nose, sinuses, teeth, mouth or other facial or cranialstructure; cranial neuralgias; and nerve trunk pain and deafferentiationpain.

In various embodiments, the a CGRP-associated disorder is diarrheaselected from the group consisting of: secretory diarrhea, osmoticdiarrhea, exudative diarrhea, motility-related diarrhea, inflammatorydiarrhea, and dysentery.

“Therapeutically effective amount” or “therapeutically effective dose”refers to that amount of the therapeutic agent being administered whichwill relieve to some extent one or more of the symptoms of the disorderbeing treated.

A therapeutically effective dose can be estimated initially from cellculture assays by determining an IC₅₀. A dose can then be formulated inanimal models to achieve a circulating plasma concentration range thatincludes the IC₅₀ as determined in cell culture. Such information can beused to more accurately determine useful doses in humans. Levels inplasma may be measured, for example, by HPLC. The exact composition,route of administration and dosage can be chosen by the individualphysician in view of the subject's condition.

Dosage regimens can be adjusted to provide the optimum desired response(e.g., a therapeutic or prophylactic response). For example, a singlebolus can be administered, several divided doses (multiple or repeat ormaintenance) can be administered over time and the dose can beproportionally reduced or increased as indicated by the exigencies ofthe therapeutic situation. It is especially advantageous to formulateparenteral compositions in dosage unit form for ease of administrationand uniformity of dosage. Dosage unit form as used herein refers tophysically discrete units suited as unitary dosages for the mammaliansubjects to be treated; each unit containing a predetermined quantity ofactive compound calculated to produce the desired therapeutic effect inassociation with the required pharmaceutical carrier. The specificationfor the dosage unit forms of the present disclosure will be dictatedprimarily by the unique characteristics of the antibody and theparticular therapeutic or prophylactic effect to be achieved.

Thus, the skilled artisan would appreciate, based upon the disclosureprovided herein, that the dose and dosing regimen is adjusted inaccordance with methods well-known in the therapeutic arts. That is, themaximum tolerable dose can be readily established, and the effectiveamount providing a detectable therapeutic benefit to a subject may alsobe determined, as can the temporal requirements for administering eachagent to provide a detectable therapeutic benefit to the subject.Accordingly, while certain dose and administration regimens areexemplified herein, these examples in no way limit the dose andadministration regimen that may be provided to a subject in practicingthe present disclosure.

It is to be noted that dosage values may vary with the type and severityof the condition to be alleviated, and may include single or multipledoses. It is to be further understood that for any particular subject,specific dosage regimens should be adjusted over time according to theindividual need and the professional judgment of the personadministering or supervising the administration of the compositions, andthat dosage ranges set forth herein are exemplary only and are notintended to limit the scope or practice of the claimed composition.Further, the dosage regimen with the compositions of this disclosure maybe based on a variety of factors, including the type of disease, theage, weight, sex, medical condition of the subject, the severity of thecondition, the route of administration, and the particular antibodyemployed. Thus, the dosage regimen can vary widely, but can bedetermined routinely using standard methods. For example, doses may beadjusted based on pharmacokinetic or pharmacodynamic parameters, whichmay include clinical effects such as toxic effects and/or laboratoryvalues. Thus, the present disclosure encompasses intra-subjectdose-escalation as determined by the skilled artisan. Determiningappropriate dosages and regimens are well-known in the relevant art andwould be understood to be encompassed by the skilled artisan onceprovided the teachings disclosed herein.

For administration to human subjects, the total monthly dose of theantibodies or antigen-binding fragments thereof of the disclosure can bein the range of 0.5-1200 mg per subject, 0.5-1100 mg per subject,0.5-1000 mg per subject, 0.5-900 mg per subject, 0.5-800 mg per subject,0.5-700 mg per subject, 0.5-600 mg per subject, 0.5-500 mg per subject,0.5-400 mg per subject, 0.5-300 mg per subject, 0.5-200 mg per subject,0.5-100 mg per subject, 0.5-50 mg per subject, 1-1200 mg per subject,1-1100 mg per subject, 1-1000 mg per subject, 1-900 mg per subject,1-800 mg per subject, 1-700 mg per subject, 1-600 mg per subject, 1-500mg per subject, 1-400 mg per subject, 1-300 mg per subject, 1-200 mg persubject, 1-100 mg per subject, or 1-50 mg per subject depending, ofcourse, on the mode of administration. For example, an intravenousmonthly dose can require about 1-1000 mg/subject. In variousembodiments, the antibodies or antigen-binding fragments thereof of thedisclosure can be administered at about 1-200 mg per subject, 1-150 mgper subject or 1-100 mg/subject. The total monthly dose can beadministered in single or divided doses and can, at the physician'sdiscretion, fall outside of the typical ranges given herein.

In various embodiments, a non-limiting daily dosing range for atherapeutically or prophylactically effective amount of an antibody orantigen-binding fragment thereof of the disclosure can be 0.001 to 100mg/kg, 0.001 to 90 mg/kg, 0.001 to 80 mg/kg, 0.001 to 70 mg/kg, 0.001 to60 mg/kg, 0.001 to 50 mg/kg, 0.001 to 40 mg/kg, 0.001 to 30 mg/kg, 0.001to 20 mg/kg, 0.001 to 10 mg/kg, 0.001 to 5 mg/kg, 0.001 to 4 mg/kg,0.001 to 3 mg/kg, 0.001 to 2 mg/kg, 0.001 to 1 mg/kg, 0.010 to 50 mg/kg,0.010 to 40 mg/kg, 0.010 to 30 mg/kg, 0.010 to 20 mg/kg, 0.010 to 10mg/kg, 0.010 to 5 mg/kg, 0.010 to 4 mg/kg, 0.010 to 3 mg/kg, 0.010 to 2mg/kg, 0.010 to 1 mg/kg, 0.1 to 50 mg/kg, 0.1 to 40 mg/kg, 0.1 to 30mg/kg, 0.1 to 20 mg/kg, 0.1 to 10 mg/kg, 0.1 to 5 mg/kg, 0.1 to 4 mg/kg,0.1 to 3 mg/kg, 0.1 to 2 mg/kg, 0.1 to 1 mg/kg, 1 to 50 mg/kg, 1 to 40mg/kg, 1 to 30 mg/kg, 1 to 20 mg/kg, 1 to 10 mg/kg, 1 to 5 mg/kg, 1 to 4mg/kg, 1 to 3 mg/kg, 1 to 2 mg/kg, or 1 to 1 mg/kg body weight.

For repeated administrations over several days or longer, depending onthe condition, the treatment is sustained until a desired suppression ofsymptoms occurs or until sufficient therapeutic levels are achieved, forexample, to reduce pain. An exemplary dosing regimen comprisesadministering an initial dose of about 2 mg/kg, followed by a weeklymaintenance dose of about 1 mg/kg of the anti-CGRP antibody, or followedby a maintenance dose of about 1 mg/kg every other week. However, otherdosage regimens may be useful, depending on the pattern ofpharmacokinetic decay that the practitioner wishes to achieve. Forexample, in some embodiments, dosing from one-four times a week iscontemplated. The progress of this therapy is easily monitored byconventional techniques and assays. The dosing regimen (including theCGRP antagonist(s) used) can vary over time. In various embodiments, theappropriate dosage of an anti-CGRP antagonist antibody will depend onthe anti-CGRP antagonist antibody (or compositions thereof) employed,the type and severity of headache (e.g., migraine) to be treated,whether the agent is administered for preventive or therapeuticpurposes, previous therapy, the patient's clinical history and responseto the agent, and the discretion of the attending physician. Typically,the clinician will administer an anti-CGRP antagonist antibody, until adosage is reached that achieves the desired result. Dose and/orfrequency can vary over course of treatment.

It is to be noted that dosage values may vary with the type and severityof the condition to be alleviated. It is to be further understood thatfor any particular subject, specific dosage regimens should be adjustedover time according to the individual need and the professional judgmentof the person administering or supervising the administration of thecompositions, and that dosage ranges set forth herein are exemplary onlyand are not intended to limit the scope or practice of the claimedcomposition.

In various embodiments, the total dose administered will achieve aplasma antibody concentration in the range of, e.g., about 1 to 1000μg/ml, about 1 to 750 μg/ml, about 1 to 500 μg/ml, about 1 to 250 μg/ml,about 10 to 1000 μg/ml, about 10 to 750 μg/ml, about 10 to 500 μg/ml,about 10 to 250 μg/ml, about 20 to 1000 μg/ml, about 20 to 750 μg/ml,about 20 to 500 μg/ml, about 20 to 250 μg/ml, about 30 to 1000 μg/ml,about 30 to 750 μg/ml, about 30 to 500 μg/ml, about 30 to 250 μg/ml.

Toxicity and therapeutic index of the pharmaceutical compositions of theinvention can be determined by standard pharmaceutical procedures incell cultures or experimental animals, e.g., for determining the LD₅₀(the dose lethal to 50% of the population) and the ED₅₀ (the dosetherapeutically effective in 50% of the population). The dose ratiobetween toxic and therapeutic effective dose is the therapeutic indexand it can be expressed as the ratio LD₅₀/ED₅₀. Compositions thatexhibit large therapeutic indices are generally preferred.

In various embodiments, single or multiple administrations of thepharmaceutical compositions are administered depending on the dosage andfrequency as required and tolerated by the subject. In any event, thecomposition should provide a sufficient quantity of at least one of theantibodies or antigen-binding fragments thereof disclosed herein toeffectively treat the subject. The dosage can be administered once butmay be applied periodically until either a therapeutic result isachieved or until side effects warrant discontinuation of therapy.

The dosing frequency of the administration of the antibody orantigen-binding fragment thereof pharmaceutical composition depends onthe nature of the therapy and the particular disease being treated. Thesubject can be treated at regular intervals, such as weekly or monthly,until a desired therapeutic result is achieved. Exemplary dosingfrequencies include, but are not limited to: once weekly without break;once weekly, every other week; once every 2 weeks; once every 3 weeks;weakly without break for 2 weeks, then monthly; weakly without break for3 weeks, then monthly; monthly; once every other month; once every threemonths; once every four months; once every five months; or once everysix months, or yearly.

Combination Therapy

As used herein, the terms “co-administration”, “co-administered” and “incombination with”, referring to the antibodies or antigen-bindingfragments thereof of the disclosure and one or more other therapeuticagents, is intended to mean, and does refer to and include thefollowing: simultaneous administration of such combination of antibodiesor antigen-binding fragments thereof of the disclosure and therapeuticagent(s) to a subject in need of treatment, when such components areformulated together into a single dosage form which releases saidcomponents at substantially the same time to said subject; substantiallysimultaneous administration of such combination of antibodies orantigen-binding fragments thereof of the disclosure and therapeuticagent(s) to a subject in need of treatment, when such components areformulated apart from each other into separate dosage forms which aretaken at substantially the same time by said subject, whereupon saidcomponents are released at substantially the same time to said subject;sequential administration of such combination of antibodies orantigen-binding fragments thereof of the disclosure and therapeuticagent(s) to a subject in need of treatment, when such components areformulated apart from each other into separate dosage forms which aretaken at consecutive times by said subject with a significant timeinterval between each administration, whereupon said components arereleased at substantially different times to said subject; andsequential administration of such combination of antibodies orantigen-binding fragments thereof of the disclosure and therapeuticagent(s) to a subject in need of treatment, when such components areformulated together into a single dosage form which releases saidcomponents in a controlled manner whereupon they are concurrently,consecutively, and/or overlappingly released at the same and/ordifferent times to said subject, where each part may be administered byeither the same or a different route.

In another aspect, the present invention relates to combinationtherapies designed to treat a CGRP-associated disorder in a subject,comprising administering to the subject a therapeutically effectiveamount of an isolated antibody or antigen-binding fragment of thepresent invention, and b) one or more additional agent useful fortreating the CGRP-associated disorder. In various embodiments, thesubject is a human subject. In various embodiments, the CGRP-associateddisorder is selected from the group consisting of headaches, hotflushes, chronic pain, type II diabetes mellitus, functional boweldisorders or an inflammatory bowel diseases, diarrhea, psoriasis, painand itch associated with arthritis and skin disease, cardiovasculardisorders, and hemodynamic derangement associated with endotoxemia,sepsis, obesity, diabetes and arthritis.

In various embodiments, the headache is selected from the groupconsisting of: migraine with or without aura; hemiplegic migraine;cluster headaches; migrainous neuralgia; chronic headaches; tensionheadaches; headaches resulting from other medical conditions (such asinfection or increased pressure in the skull due to a tumor); chronicparoxysmal hemicrania; miscellaneous headache unassociated with astructural lesion; headache associated with a non-vascular intracranialdisorder; headache associated with the administration of a substance orits withdrawal; headache associated with noncephalic infection; headacheassociated with a metabolic disorder; headache associated with adisorder of the cranium, neck, eyes, ears, nose, sinuses, teeth, mouthor other facial or cranial structure; cranial neuralgias; and nervetrunk pain and deafferentiation pain.

In various embodiments, the CGRP-associated disorder is headache and theone or more additional agent is selected from the group consisting of:5-HT1-like agonists (and agonists acting at other 5-HT1 sites), andnon-steroidal anti-inflammatory drugs (NSAIDs). In various embodiments,the 5-HT1 agonist is a triptan selected from the group consisting ofsumatriptan, zolmitriptan, naratriptan, rizatriptan, eletriptan,almotriptan, and frovatriptan. In various embodiments, the one or moreadditional agent is a NSAID selected from the group consisting ofnaproxen, flurbiprofen, ketoprofen, oxaprozin, etodolac, indomethacin,ketorolac, nabumetone, mefanamic acid, and piroxican. In variousembodiments, the one or more additional agent is a NSAID is acyclooxygenase-2 (COX-2) inhibitor selected from the group consistingof: celecoxib; rofecoxib; meloxicam; JTE-522; L-745,337; NS398; andpharmaceutically acceptable salts thereof. In various embodiments, theone or more additional agent is an ergot alkaloid selected from thegroup consisting of ergotamine tartrate, ergonovine maleate, andergoloid mesylates (e.g., dihydroergocornine, dihydroergocristine,dihydroergocryptine, and dihydroergotamine mesylate (DHE 45)). Invarious embodiments, a synergy exists between the isolated antibody orantigen-binding fragment and the additional therapies whenco-administered.

In various embodiments, the CGRP-associated disorder is hot flashes andthe one or more additional agent includes, but are not limited to,hormone-based treatments, including estrogens and/or progestins.

In various embodiments, the combination therapy comprises administeringthe antibody or antigen-binding fragment thereof and the one or moreadditional therapies simultaneously. In various embodiments, antibody orantigen-binding fragment thereof composition and the one or moreadditional therapies are administered sequentially, i.e., the antibodyor antigen-binding fragment thereof composition is administered eitherprior to or after the administration of the one or more additionaltherapies.

In various embodiments, the administrations of the antibody orantigen-binding fragment thereof composition and the one or moreadditional therapies are concurrent, i.e., the administration period ofthe antibody or antigen-binding fragment thereof composition and the oneor more additional therapies overlap with each other.

In various embodiments, the administrations of the antibody orantigen-binding fragment thereof composition and the one or moreadditional therapies are non-concurrent. For example, in variousembodiments, the administration of the antibody or antigen-bindingfragment thereof composition is terminated before the one or moreadditional therapies is administered. In various embodiments, theadministration of the one or more additional therapies is terminatedbefore the antibody or antigen-binding fragment thereof composition isadministered.

When the antibody or antigen-binding fragment thereof disclosed hereinis administered in combination with one or more additional therapies,either concomitantly or sequentially, such antibody or antigen-bindingfragment thereof may enhance the therapeutic effect of the one or moreadditional therapies or overcome cellular resistance to the one or moreadditional therapies. This allows for decreased dosage or duration ofthe one or more additional therapies, thereby reducing the undesirableside effects, or restores the effectiveness of the one or moreadditional therapies.

Diagnostic Uses

In another aspect, the present invention provides a method for detectingin vitro or in vivo the presence of human CGRP peptide in a sample,e.g., for diagnosing a human CGRP-related disorder. In some methods,this is achieved by contacting a sample to be tested, along with acontrol sample, with a human sequence antibody or a human monoclonalantibody of the invention, or an antigen-binding portion thereof (or abispecific or multispecific molecule), under conditions that allow forformation of a complex between the antibody and human CGRP. Complexformation is then detected (e.g., using an ELISA) in both samples, andany statistically significant difference in the formation of complexesbetween the samples is indicative the presence of human CGRP antigen inthe test sample.

In various embodiments, methods are provided for detecting aCGRP-related disorder or confirming the diagnosis of a CGRP-relateddisorder in a subject. The method includes contacting a biologicalsample from the subject with an isolated antibody or antigen-bidingfragment thereof of the invention and detecting binding of the isolatedhuman monoclonal antibody or antigen-binding fragment thereof to thesample. An increase in binding of the isolated human monoclonal antibodyor antigen-binding fragment thereof to the sample as compared to bindingof the isolated human monoclonal antibody or antigen-binding fragmentthereof to a control sample detects a CGRP-related disorder in thesubject or confirms the diagnosis of a CGRP-related disorder in thesubject. The control can be a sample from a subject known not to have aCGRP-related disorder, or a standard value. The sample can be anysample, including, but not limited to, tissue from biopsies, autopsiesand pathology specimens. Biological samples also include sections oftissues, for example, frozen sections taken for histological purposes.Biological samples further include body fluids, such as blood, serum,plasma, sputum, and spinal fluid.

In one embodiment, a kit is provided for detecting CGRP in a biologicalsample, such as a blood sample. Kits for detecting a polypeptide willtypically comprise a human antibody that specifically binds CGRP, suchas any of the antibodies disclosed herein. In some embodiments, anantibody fragment, such as an Fv fragment is included in the kit. For invivo uses, the antibody can be a scFv fragment. In a further embodiment,the antibody is labeled (for example, with a fluorescent, radioactive,or an enzymatic label).

In one embodiment, a kit includes instructional materials disclosingmeans of use of an antibody that specifically binds CGRP. Theinstructional materials may be written, in an electronic form (such as acomputer diskette or compact disk) or may be visual (such as videofiles). The kits may also include additional components to facilitatethe particular application for which the kit is designed. Thus, forexample, the kit may additionally contain means of detecting a label(such as enzyme substrates for enzymatic labels, filter sets to detectfluorescent labels, appropriate secondary labels such as a secondaryantibody, or the like). The kits may additionally include buffers andother reagents routinely used for the practice of a particular method.Such kits and appropriate contents are well known to those of skill inthe art.

In one embodiment, the diagnostic kit comprises an immunoassay. Althoughthe details of the immunoassays may vary with the particular formatemployed, the method of detecting CGRP in a biological sample generallyincludes the steps of contacting the biological sample with an antibodywhich specifically reacts, under immunologically reactive conditions, toCGRP. The antibody is allowed to specifically bind under immunologicallyreactive conditions to form an immune complex, and the presence of theimmune complex (bound antibody) is detected directly or indirectly.

In various embodiments, the antibodies or antigen-binding fragments canbe labeled or unlabeled for diagnostic purposes. Typically, diagnosticassays entail detecting the formation of a complex resulting from thebinding of an antibody to CGRP. The antibodies can be directly labeled.A variety of labels can be employed, including, but not limited to,radionuclides, fluorescers, enzymes, enzyme substrates, enzymecofactors, enzyme inhibitors and ligands (e.g., biotin, haptens).Numerous appropriate immunoassays are known to the skilled artisan (see,for example, U.S. Pat. Nos. 3,817,827; 3,850,752; 3,901,654; and4,098,876). When unlabeled, the antibodies can be used in assays, suchas agglutination assays. Unlabeled antibodies can also be used incombination with another (one or more) suitable reagent which can beused to detect antibody, such as a labeled antibody (e.g., a secondantibody) reactive with the first antibody (e.g., anti-idiotypeantibodies or other antibodies that are specific for the unlabeledimmunoglobulin) or other suitable reagent (e.g., labeled protein A).

The antibody or antigen-binding fragment provided herein may also beused in a method of detecting the susceptibility of a mammal to certaindiseases. To illustrate, the method can be used to detect thesusceptibility of a mammal to diseases which progress based on theamount of CGRP present on cells and/or the number of CGRP-positive cellsin a mammal. In one embodiment, the application provides a method ofdetecting susceptibility of a mammal to a tumor. In this embodiment, asample to be tested is contacted with an antibody which binds to CGRP orportion thereof under conditions appropriate for binding of saidantibody thereto, wherein the sample comprises cells which express CGRPin normal individuals. The binding of antibody and/or amount of bindingis detected, which indicates the susceptibility of the individual to atumor, wherein higher levels of receptor correlate with increasedsusceptibility of the individual to a tumor.

In various embodiments, the antibodies or antigen-binding fragments areattached to a label that is able to be detected (e.g., the label can bea radioisotope, fluorescent compound, enzyme or enzyme co-factor). Theactive moiety may be a radioactive agent, such as: radioactive heavymetals such as iron chelates, radioactive chelates of gadolinium ormanganese, positron emitters of oxygen, nitrogen, iron, carbon, orgallium, ⁴³K, ⁵²Fe, ⁵⁷Co, ⁶⁷Cu, ⁶⁷Ga, ⁶⁸Ga, ¹²³I, ¹²⁵I, ¹³¹I, ¹³²I, or⁹⁹Tc. A binding agent affixed to such a moiety may be used as an imagingagent and is administered in an amount effective for diagnostic use in amammal such as a human and the localization and accumulation of theimaging agent is then detected. The localization and accumulation of theimaging agent may be detected by radioscintigraphy, nuclear magneticresonance imaging, computed tomography or positron emission tomography.

Immunoscintigraphy using antibodies or antigen-binding fragmentsdirected at CGRP may be used to detect and/or diagnose cancers andvasculature. For example, monoclonal antibodies against the CGRP markerlabeled with ⁹⁹Technetium, ¹¹¹Indium, or ¹²⁵Iodine may be effectivelyused for such imaging. As will be evident to the skilled artisan, theamount of radioisotope to be administered is dependent upon theradioisotope. Those having ordinary skill in the art can readilyformulate the amount of the imaging agent to be administered based uponthe specific activity and energy of a given radionuclide used as theactive moiety. Typically 0.1-100 millicuries per dose of imaging agent,or 1-10 millicuries, or 2-5 millicuries are administered. Thus, thecompositions disclosed are useful as imaging agents comprising atargeting moiety conjugated to a radioactive moiety comprise 0.1-100millicuries, in some embodiments 1-10 millicuries, in some embodiments2-5 millicuries, in some embodiments 1-5 millicuries.

Bispecific Molecules

In another aspect, the present invention features bispecific moleculescomprising an anti-CGRP antibody, or antigen-binding fragment thereof,of the invention. An antibody of the invention, or antigen-bindingfragment thereof, can be derivatized or linked to another functionalmolecule, e.g., another peptide or protein (e.g., another antibody orligand for a receptor) to generate a bispecific molecule that binds toat least two different binding sites or target molecules. The antibodyof the invention may in fact be derivatized or linked to more than oneother functional molecule to generate multispecific molecules that bindto more than two different binding sites and/or target molecules; suchmultispecific molecules are also intended to be encompassed by the term“bispecific molecule” as used herein. To create a bispecific molecule ofthe invention, an antibody of the invention can be functionally linked(e.g., by chemical coupling, genetic fusion, noncovalent association orotherwise) to one or more other binding molecules, such as anotherantibody, antibody fragment, peptide or binding mimetic, such that abispecific molecule results. In various embodiments, the inventionincludes bispecific molecules capable of binding both to FcγR or FcαRexpressing effector cells (e.g., monocytes, macrophages orpolymorphonuclear cells (PMNs)), and to target cells expressing PD. Insuch embodiments, the bispecific molecules target CGRP expressing cellsto effector cell and trigger Fc receptor-mediated effector cellactivities, e.g., phagocytosis of an CGRP expressing cells, antibodydependent cell-mediated cytotoxicity (ADCC), cytokine release, orgeneration of superoxide anion. Methods of preparing the bispecificmolecules of the present invention are well known in the art.

Polynucleotides and Antibody Expression

The application further provides polynucleotides comprising a nucleotidesequence encoding an anti-CGRP antibody or antigen-binding fragmentthereof. Because of the degeneracy of the genetic code, a variety ofnucleic acid sequences encode each antibody amino acid sequence. Theapplication further provides polynucleotides that hybridize understringent or lower stringency hybridization conditions, e.g., as definedherein, to polynucleotides that encode an antibody that binds to humanCGRP.

Stringent hybridization conditions include, but are not limited to,hybridization to filter-bound DNA in 6×SSC at about 45° C. followed byone or more washes in 0.2×SSC/0.1% SDS at about 50-65° C., highlystringent conditions such as hybridization to filter-bound DNA in 6×SSCat about 45° C. followed by one or more washes in 0.1×SSC/0.2% SDS atabout 60° C., or any other stringent hybridization conditions known tothose skilled in the art (see, for example, Ausubel, F. M. et al., eds.1989 Current Protocols in Molecular Biology, vol. 1, Green PublishingAssociates, Inc. and John Wiley and Sons, Inc., NY at pages 6.3.1 to6.3.6 and 2.10.3).

The polynucleotides may be obtained, and the nucleotide sequence of thepolynucleotides determined, by any method known in the art. For example,if the nucleotide sequence of the antibody is known, a polynucleotideencoding the antibody may be assembled from chemically synthesizedoligonucleotides (e.g., as described in Kutmeier et al., BioTechniques17:242 (1994)), which, briefly, involves the synthesis of overlappingoligonucleotides containing portions of the sequence encoding theantibody, annealing and ligating of those oligonucleotides, and thenamplification of the ligated oligonucleotides by PCR. In one embodiment,the codons that are used comprise those that are typical for human ormouse (see, e.g., Nakamura, Y., Nucleic Acids Res. 28: 292 (2000)).

A polynucleotide encoding an antibody may also be generated from nucleicacid from a suitable source. If a clone containing a nucleic acidencoding a particular antibody is not available, but the sequence of theantibody molecule is known, a nucleic acid encoding the immunoglobulinmay be chemically synthesized or obtained from a suitable source (e.g.,an antibody cDNA library, or a cDNA library generated from, or nucleicacid, preferably polyA+RNA, isolated from, any tissue or cellsexpressing the antibody, such as hybridoma cells selected to express anantibody) by PCR amplification using synthetic primers hybridizable tothe 3′ and 5′ ends of the sequence or by cloning using anoligonucleotide probe specific for the particular gene sequence toidentify, e.g., a cDNA clone from a cDNA library that encodes theantibody. Amplified nucleic acids generated by PCR may then be clonedinto replicable cloning vectors using any method well known in the art.

The present invention is also directed to host cells that express a CGRPand/or the anti-CGRP antibodies of the invention. A wide variety of hostexpression systems known in the art can be used to express an antibodyof the present invention including prokaryotic (bacterial) andeukaryotic expression systems (such as yeast, baculovirus, plant,mammalian and other animal cells, transgenic animals, and hybridomacells), as well as phage display expression systems.

An antibody of the invention can be prepared by recombinant expressionof immunoglobulin light and heavy chain genes in a host cell. To expressan antibody recombinantly, a host cell is transformed, transduced,infected or the like with one or more recombinant expression vectorscarrying DNA fragments encoding the immunoglobulin light and/or heavychains of the antibody such that the light and/or heavy chains areexpressed in the host cell. The heavy chain and the light chain may beexpressed independently from different promoters to which they areoperably-linked in one vector or, alternatively, the heavy chain and thelight chain may be expressed independently from different promoters towhich they are operably-linked in two vectors one expressing the heavychain and one expressing the light chain. Optionally, the heavy chainand light chain may be expressed in different host cells.

Additionally, the recombinant expression vector can encode a signalpeptide that facilitates secretion of the antibody light and/or heavychain from a host cell. The antibody light and/or heavy chain gene canbe cloned into the vector such that the signal peptide isoperably-linked in-frame to the amino terminus of the antibody chaingene. The signal peptide can be an immunoglobulin signal peptide or aheterologous signal peptide. Preferably, the recombinant antibodies aresecreted into the medium in which the host cells are cultured, fromwhich the antibodies can be recovered or purified.

An isolated DNA encoding a HCVR can be converted to a full-length heavychain gene by operably-linking the HCVR-encoding DNA to another DNAmolecule encoding heavy chain constant regions. The sequences of human,as well as other mammalian, heavy chain constant region genes are knownin the art. DNA fragments encompassing these regions can be obtainede.g., by standard PCR amplification. The heavy chain constant region canbe of any type, (e.g., IgG, IgA, IgE, IgM or IgD), class (e.g., IgG₁,IgG₂, IgG₃ and IgG₄) or subclass constant region and any allotypicvariant thereof as described in Kabat (supra).

An isolated DNA encoding a LCVR region may be converted to a full-lengthlight chain gene (as well as to a Fab light chain gene) by operablylinking the LCVR-encoding DNA to another DNA molecule encoding a lightchain constant region. The sequences of human, as well as othermammalian, light chain constant region genes are known in the art. DNAfragments encompassing these regions can be obtained by standard PCRamplification. The light chain constant region can be a kappa or lambdaconstant region.

In addition to the antibody heavy and/or light chain gene(s), arecombinant expression vector of the invention carries regulatorysequences that control the expression of the antibody chain gene(s) in ahost cell. The term “regulatory sequence” is intended to includepromoters, enhancers and other expression control elements (e.g.,polyadenylation signals), as needed, that control the transcription ortranslation of the antibody chain gene(s). The design of the expressionvector, including the selection of regulatory sequences may depend onsuch factors as the choice of the host cell to be transformed, the levelof expression of protein desired. Preferred regulatory sequences formammalian host cell expression include viral elements that direct highlevels of protein expression in mammalian cells, such as promotersand/or enhancers derived from cytomegalovirus (CMV), Simian Virus 40(SV40), adenovirus, (e.g., the adenovirus major late promoter (AdMLP))and/or polyoma virus.

Additionally, the recombinant expression vectors of the invention maycarry additional sequences, such as sequences that regulate replicationof the vector in host cells (e.g., origins of replication) and one ormore selectable marker genes. The selectable marker gene facilitatesselection of host cells into which the vector has been introduced. Forexample, typically the selectable marker gene confers resistance todrugs, such as G418, hygromycin, or methotrexate, on a host cell intowhich the vector has been introduced. Preferred selectable marker genesinclude the dihydrofolate reductase (dhfr) gene (for use in dhfr-minushost cells with methotrexate selection/amplification), the neo gene (forG418 selection), and glutamine synthetase (GS) in a GS-negative cellline (such as NSO) for selection/amplification.

For expression of the light and/or heavy chains, the expressionvector(s) encoding the heavy and/or light chains is introduced into ahost cell by standard techniques e.g. electroporation, calcium phosphateprecipitation, DEAE-dextran transfection, transduction, infection andthe like. Although it is theoretically possible to express theantibodies of the invention in either prokaryotic or eukaryotic hostcells, eukaryotic cells are preferred, and most preferably mammalianhost cells, because such cells are more likely to assemble and secrete aproperly folded and immunologically active antibody. Preferred mammalianhost cells for expressing the recombinant antibodies of the inventioninclude Chinese Hamster Ovary (CHO cells) [including dhfr minus CHOcells, as described in Urlaub and Chasin, Proc. Natl. Acad. Sci. USA77:4216-20, 1980, used with a DHFR selectable marker, e.g. as describedin Kaufman and Sharp, J. Mol. Biol. 159:601-21, 1982], NSO myelomacells, COS cells, and SP2/0 cells. When recombinant expression vectorsencoding antibody genes are introduced into mammalian host cells, theantibodies are produced by culturing the host cells for a period of timesufficient to allow for expression of the antibody in the host cells or,more preferably, secretion of the antibody into the culture medium inwhich the host cells are grown under appropriate conditions known in theart. Antibodies can be recovered from the host cell and/or the culturemedium using standard purification methods.

The invention provides a host cell comprising a nucleic acid molecule ofthe present invention. Preferably a host cell of the invention comprisesone or more vectors or constructs comprising a nucleic acid molecule ofthe present invention. For example, a host cell of the invention is acell into which a vector of the invention has been introduced, saidvector comprising a polynucleotide encoding a LCVR of an antibody of theinvention and/or a polynucleotide encoding a HCVR of the invention. Theinvention also provides a host cell into which two vectors of theinvention have been introduced; one comprising a polynucleotide encodinga LCVR of an antibody of the invention and one comprising apolynucleotide encoding a HCVR present in an antibody of the inventionand each operably-linked to enhancer/promoter regulatory elements (e.g.,derived from SV40, CMV, adenovirus and the like, such as a CMVenhancer/AdMLP promoter regulatory element or an SV40 enhancer/AdMLPpromoter regulatory element) to drive high levels of transcription ofthe genes.

Once expressed, the intact antibodies, individual light and heavychains, or other immunoglobulin forms of the present invention can bepurified according to standard procedures of the art, including ammoniumsulfate precipitation, ion exchange, affinity (e.g., Protein A), reversephase, hydrophobic interaction column chromatography, hydroxyapatitechromatography, gel electrophoresis, and the like. Standard proceduresfor purification of therapeutic antibodies are described, for example,by Feng L1, Joe X. Zhou, Xiaoming Yang, Tim Tressel, and Brian Lee in anarticle entitled “Current Therapeutic Antibody Production and ProcessOptimization” (BioProcessing Journal, September/October2005)(incorporated by reference in its entirety for purposes of teachingpurification of therapeutic antibodies). Additionally, standardtechniques for removing viruses from recombinantly expressed antibodypreparations are also known in the art (see, for example, Gerd Kern andMani Krishnan, “Viral Removal by Filtration: Points to Consider”(Biopharm International, October 2006)). The effectiveness of filtrationto remove viruses from preparations of therapeutic antibodies is knownto be at least in part dependent on the concentration of protein and/orthe antibody in the solution to be filtered. The purification processfor antibodies of the present invention may include a step of filteringto remove viruses from the mainstream of one or more chromatographyoperations. Preferably, prior to filtering through a pharmaceuticalgrade nanofilter to remove viruses, a chromatography mainstreamcontaining an antibody of the present invention is diluted orconcentrated to give total protein and/or total antibody concentrationof about 1 g/L to about 3 g/L. Even more preferably, the nanofilter is aDV20 nanofilter (e.g., Pall Corporation; East Hills, N.Y.).Substantially pure immunoglobulins of at least about 90%, about 92%,about 94% or about 96% homogeneity are preferred, and about 98 to about99% or more homogeneity most preferred, for pharmaceutical uses. Oncepurified, partially or to homogeneity as desired, the sterile antibodiesmay then be used therapeutically, as directed herein.

In view of the aforementioned discussion, the present invention isfurther directed to an antibody obtainable by a process comprising thesteps of culturing a host cell including, but not limited to amammalian, plant, bacterial, transgenic animal, or transgenic plant cellwhich has been transformed by a polynucleotide or a vector comprisingnucleic acid molecules encoding antibodies of the invention so that thenucleic acid is expressed and, optionally, recovering the antibody fromthe host cell culture medium.

In certain aspects, the present application provides hybridoma celllines, as well as to the monoclonal antibodies produced by thesehybridoma cell lines. The cell lines disclosed have uses other than forthe production of the monoclonal antibodies. For example, the cell linescan be fused with other cells (such as suitably drug-marked humanmyeloma, mouse myeloma, human-mouse heteromyeloma or humanlymphoblastoid cells) to produce additional hybridomas, and thus providefor the transfer of the genes encoding the monoclonal antibodies. Inaddition, the cell lines can be used as a source of nucleic acidsencoding the anti-CGRP immunoglobulin chains, which can be isolated andexpressed (e.g., upon transfer to other cells using any suitabletechnique (see e.g., Cabilly et al., U.S. Pat. No. 4,816,567; Winter,U.S. Pat. No. 5,225,539)). For instance, clones comprising a rearrangedanti-CGRP light or heavy chain can be isolated (e.g., by PCR) or cDNAlibraries can be prepared from mRNA isolated from the cell lines, andcDNA clones encoding an anti-CGRP immunoglobulin chain can be isolated.Thus, nucleic acids encoding the heavy and/or light chains of theantibodies or portions thereof can be obtained and used in accordancewith recombinant DNA techniques for the production of the specificimmunoglobulin, immunoglobulin chain, or variants thereof (e.g.,humanized immunoglobulins) in a variety of host T-cells or in an invitro translation system. For example, the nucleic acids, includingcDNAs, or derivatives thereof encoding variants such as a humanizedimmunoglobulin or immunoglobulin chain, can be placed into suitableprokaryotic or eukaryotic vectors (e.g., expression vectors) andintroduced into a suitable host T-cell by an appropriate method (e.g.,transformation, transfection, electroporation, infection), such that thenucleic acid is operably linked to one or more expression controlelements (e.g., in the vector or integrated into the host T-cellgenome). For production, host T-cells can be maintained under conditionssuitable for expression (e.g., in the presence of inducer, suitablemedia supplemented with appropriate salts, growth factors, antibiotic,nutritional supplements, etc.), whereby the encoded polypeptide isproduced. If desired, the encoded protein can be recovered and/orisolated (e.g., from the host T-cells or medium). It will be appreciatedthat the method of production encompasses expression in a host T-cell ofa transgenic animal (see e.g., WO 92/03918, GenPharm International,published Mar. 19, 1992)(incorporated by reference in its entirety).

Host cells can also be used to produce portions, or fragments, of intactantibodies, e.g., Fab fragments or scFv molecules by techniques that areconventional. For example, it may be desirable to transfect a host cellwith DNA encoding either the light chain or the heavy chain of anantibody of this invention. Recombinant DNA technology may also be usedto remove some or all the DNA encoding either or both of the light andheavy chains that is not necessary for binding to human CGRP. Themolecules expressed from such truncated DNA molecules are alsoencompassed by the antibodies of the invention.

Methods for expression of single chain antibodies and/or refolding to anappropriate active form, including single chain antibodies, frombacteria such as E. coli have been described and are well-known and areapplicable to the antibodies disclosed herein (see, e.g., Buchner etal., Anal. Biochem. 205:263-270, 1992; Pluckthun, Biotechnology 9:545,1991; Huse et al., Science 246:1275, 1989 and Ward et al., Nature341:544, 1989, all incorporated by reference herein).

Often, functional heterologous proteins from E. coli or other bacteriaare isolated from inclusion bodies and require solubilization usingstrong denaturants, and subsequent refolding. During the solubilizationstep, as is well known in the art, a reducing agent must be present toseparate disulfide bonds. An exemplary buffer with a reducing agent is:0.1 M Tris pH 8, 6 M guanidine, 2 mM EDTA, 0.3 M DTE (dithioerythritol).Reoxidation of the disulfide bonds can occur in the presence of lowmolecular weight thiol reagents in reduced and oxidized form, asdescribed in Saxena et al., Biochemistry 9: 5015-5021, 1970,incorporated by reference herein, and especially as described by Buchneret al., supra.

Renaturation is typically accomplished by dilution (for example,100-fold) of the denatured and reduced protein into refolding buffer. Anexemplary buffer is 0.1 M Tris, pH 8.0, 0.5 M L-arginine, 8 mM oxidizedglutathione (GSSG), and 2 mM EDTA.

As a modification to the two chain antibody purification protocol, theheavy and light chain regions are separately solubilized and reduced andthen combined in the refolding solution. An exemplary yield is obtainedwhen these two proteins are mixed in a molar ratio such that a 5 foldmolar excess of one protein over the other is not exceeded. Excessoxidized glutathione or other oxidizing low molecular weight compoundscan be added to the refolding solution after the redox-shuffling iscompleted.

In addition to recombinant methods, the antibodies, labeled antibodiesand antigen-binding fragments thereof that are disclosed herein can alsobe constructed in whole or in part using standard peptide synthesis.Solid phase synthesis of the polypeptides of less than about 50 aminoacids in length can be accomplished by attaching the C-terminal aminoacid of the sequence to an insoluble support followed by sequentialaddition of the remaining amino acids in the sequence. Techniques forsolid phase synthesis are described by Barany & Merrifield, ThePeptides: Analysis, Synthesis, Biology. Vol. 2: Special Methods inPeptide Synthesis, Part A. pp. 3-284; Merrifield et al., J. Am. Chem.Soc. 85:2149-2156, 1963, and Stewart et al., Solid Phase PeptideSynthesis, 2nd ed., Pierce Chem. Co., Rockford, Ill., 1984. Proteins ofgreater length may be synthesized by condensation of the amino andcarboxyl termini of shorter fragments. Methods of forming peptide bondsby activation of a carboxyl terminal end (such as by the use of thecoupling reagent N,N′-dicylohexylcarbodimide) are well known in the art.

The following examples are offered to more fully illustrate theinvention, but are not construed as limiting the scope thereof.

Example 1 Generation of Monoclonal Antibodies Targeting Specifically toHuman CGRP

Female Balb/c and C57Bl/6 mouse were immunized three times (every secondweek). For the 1^(st) immunization, the mice were immunizedsubcutaneously with 50 μg of CGRP protein (R&D Systems Cat #3012) permouse. Antigen was injected as 1:1 mixture with Complete Freund'sAdjuvant (Sigma, St. Louis, Mo.). For the 2^(nd) and 3rd immunizations,the mice were immunized intraperitoneally with 25 μg of CGRP protein permouse. Antigen was injected as 1:1 mixture with Incomplete Freund'sAdjuvant (Sigma, St. Louis, Mo.) in the second and third doses. Micewere given a final boost intraperitoneally with 25 μg of CGRP, andsplenocytes were harvested 4 days later for fusion with myeloma cellline NSO from ATCC (Allendale, N.J.). Electric fusion methods are usedto obtain hybridoma cells and then the hybridoma supernatants arescreened for antigen binding, ligand blocking, IgG binning, referenceantibody binding, and FACS binding. 20 murine mAbs were ultimatelyselected from the initial screens for subcloning (limited dilutionmethod). BD Cell MAb Medium was used to grow hybridomas in rollerbottles for the collection of supernatants for antibody production. mAbswere purified with Protein A affinity chromatography. Estimated purityof mAbs was higher than 90% based on SDS-PAGE Coomassie staining. 15purified mAbs (6G5A6B9, 37B1F8B3, 37H5E2H9, 37H5C2D2, 30B8G9B11,6G5D2B1, 30B8F4C8, 24G7B5G11, 24G7G12C7, 37B1B5B3, 37H5G5E1, 37B1 D4D7,6G5C8C4, 24G7B10G2, 24G7B7B12) were selected for secondary screeningwhich comprised: human CGRP binding avidity assays (ELISA), murine CGRPcrossreactivity assays (ELISA), primate CGRP crossreactivity assays(ELISA), SK-N-MC cells CGRP assay, and epitope binning screening.

The CGRP receptor is a hetero-trimeric complex consisting of theCalcitonin receptor-Like Receptor (CLR, a G-protein coupled receptor)and Receptor Activity Modifying Protein (RAMP)-1, coupledcytoplasmically to Receptor Component Protein (RCP). The binding of CGRPto its receptor results in the stimulation of cAMP production. The humanneuroepithelioma cell line SK-N-MC naturally expresses the CGRP receptorand can be used to assess whether anti-CGRP antibodies are able toinhibit CGRP-induced cAMP production (RJ Benschop et al., Osteoarthritisand Cartilage, 22:578-585, 2014; Shi et al., J Pharmacol Exp Ther.,356:223-231, 2016). The SK-N-MC cell line was used to assess the effectsof the 15 purified mAbs (6G5A6B9, 37B1F8B3, 37H5E2H9, 37H5C2D2,30B8G9B11, 6G5D2B1, 30B8F4C8, 24G7B5G11, 24G7G12C7, 37616563, 37H5G5E1,37B1 D4D7, 6G5C8C4, 24G7B10G2, 24G7B7B12). Briefly, the 15 purified Absare preincubated with 30 pM CGRP for 30 minutes at room temp and thendiluted 2-fold into SK-N-MC cells and incubated 30 minutes at 37° C. ina 5% CO2 incubator. 25 μL of cAMP-d2/lysis buffer and 25 μL ofanti-cAMP-cryptate/lysis buffer is then added to each well. The platesare sealed and incubated at 4° C. for 1 hour. The SK-N-MC cell assaymedium contains fixed concentrations of CGRP (100 pM) during the assayperiod. Thei inhibitory effect is detected as a reduction of CGRPrelative to control. The OD665/620 ratio as a measure of cAMP levels wasdetermined in a Perkin Elmer Envision.

The secondary assay data for the 15 murine mAbs is summarized in FIGS. 1and 2 and in Table 3:

TABLE 3 Primate Rodent Human CGRP CGRP CGRP Binding Binding BindingSK-N-MC Murine ELISA ELISA ELISA CGRP mAb EC50 (nM) EC50 (nM) EC50 (nM)IC50 (pM) 6G5D2B1 0.33 1.015 0.390 241 6G5A6B9 0.152 0.508 0.150  926G5C8C4 0.101 0.361 0.100 101 24G7B7B12 0.157 0.827 0.160 ND 24G7B10G20.072 0.328 0.080 195 24G7B5G11 0.069 0.313 0.060 326 24G7G12C7 0.5041.559 0.600 ND 30B8G9B11 0.070 0.296 0.070 152 30B8F4C8 0.078 0.3620.080 130 37B1F8B3 0.068 0.274 0.060 160 37B1B5B3 0.057 0.320 0.060 20237B1D4D7 0.057 0.345 0.060 110 37H5E2H9 0.077 0.352 0.080 171 37H5C2D20.082 0.406 0.080 435 37H5G5E1 0.052 1.025 0.050 177As depicted in Table 3, the anti-CGRP murine monoclonal antibodies bindhuman and macaque CGRP with high affinity and inhibit the production ofcAMP in the SK-N-MC assay with 1050 between 92-435 pM as required for aclinical mAb therapeutic.

Based on the cumulative results of the secondary assays, purified murinemAbs 6G5C8C4 (“A1”), 24G7B10G2 (“A2”) and 30B8G9B11 (“A3”) were selectedfor sequencing and further analysis. Total RNA was extracted from frozenhybridoma cells following the technical manual of TRIzol® Reagent. Thetotal RNA was analyzed by agarose gel electrophoresis. Total RNA wasreverse transcribed into cDNA using isotype-specific anti-sense primersor universal primers following the technical manual of PrimeScript™ 1 stStrand cDNA Synthesis Kit. PCR was then performed to amplify thevariable regions (heavy and light chains) of the antibodies, which werethen cloned into a standard cloning vector separately and sequenced.Murine mAbs 6G5C8C4 (“A1”), 24G7B10G2 (“A2”) and 30B8G9B11 (“A3”) areall IgG2a, k isotype antibodies from epitope bin 1 and comprise theheavy chain variable region sequences set forth in SEQ ID NOs: 17, 19and 21, respectively, and the light chain variable region sequences setforth in SEQ ID NOs: 23, 25 and 27, respectively. The heavy chainvariable regions of mAbs 6G5C8C4 (“A1”), 24G7B10G2 (“A2”) and 30B8G9B11(“A3”) are encoded by the nucleic acid sequences set forth in SEQ IDNOs: 18, 20 and 22, respectively, and the light chain variable regionsof mAbs 6G5C8C4 (“A1”), 24G7B10G2 (“A2”) and 30B8G9B11 (“A3”) areencoded by the nucleic acid sequences set forth in SEQ ID NOs: 24, 26and 28, respectively.

Example 2 Generation of a Chimeric IgG4 Targeting Human CGRP

Using the HCVR and LCVR sequences of mAb 30B8G9B11 (“A3”), amurine-human IgG4 chimeric Fab (hereinafter “chimeric IgG4”) wasprepared which comprised the heavy chain sequence set forth in SEQ IDNO: 29 and the light chain sequence set forth in SEQ ID NO: 31. Theheavy chain and light chain of the chimeric IgG4 are encoded by thenucleic acids set forth in SEQ ID NOs: 30 and 32, respectively. Nucleicacids encded by SEQ ID NO: 30 and SEQ ID NO: 32 which include a leadersequence were amplified and inserted into pTT5 to make an expressionplasmid of the full-length chimeric IgG4. The heavy chain and lightchain expression plasmids were used to co-transfect 100 mL HEK293-6Ecells. The recombinant IgG4 secreted into to media was purified usingprotein A affinity. The purified antibody was buffer-exchanged into PBSusing PD-10 desalting column. The purified chimeric IgG4 migrated as˜170 kDa band in SDS-PAGE under non-reducing conditions, ˜55 kDa and ˜30kDA bands under reducing condition. The purity of the chimeric IgG4is >85%, and the yield form the 100 mL culture was ˜2.4 mg/L.

The binding affinity between the chimeric IgG4 to antigen CGRP wasdetermined using a Surface Plasmon Resonance (SPR) biosensor, BiacoreT200 (GE Healthcare). 30B8G9B11 (“A3”) and chimeric IgG4 were eachimmobilized on the sensor chip through an amine coupling method. AntigenCGRP protein was used as analyte. The data of dissociation (k_(d)) andassociation (k_(a)) rate constants were obtained using Biacore T200evaluation software. The equilibrium dissociation constants (K_(D)) werecalculated from the ratio of k_(d) over k_(a). The results aresummarized in Table 4:

TABLE 4 k_(a) Rmax Chi² Ligand Analyte (1/Ms) k_(d) (1/s) K_(D) (M) (RU)(RU²) Chimeric CGRP 6.28E+06 1.51E−05 2.40E−12 40.61 0.0119 IgG4 Murine5.33E+06 1.56E−05 2.92E−12 28.54 0.015  A3

Example 3 Generation of Humanized Abs Specifically Targeting Human CGRP

A CDR grafting and back mutation method was used to prepare humanizedanti-CGRP mAbs derived from murine mAb 30B8G9B11 (“A3”). Briefly, theCDRs of parental murine antibody A3 were grafted into the humanacceptors to obtain humanized light chains and humanized heavy chainsfor the parental antibody. Human acceptors selected for VH and VL wereGen Bank AAP97932.1 and AKU38886.1, respectively. The CDRs and HV loopsof the human acceptors were replaced by their mouse counterparts (CDRgrafting), which gave the sequence of the grafted antibody, identifiedhereinafter as “CGRP-grafted Ab”, which comprises the heavy chainvariable region sequence set forth in SEQ ID NO: 60 and the light chainvariable region sequence set forth in SEQ ID NO: 61.

Canonical residues in CDR, framework region and residues on VH-VLinterface in the grafted antibody that are believed to be important forthe binding activity were selected for replacement with parentalantibody counterparts. Homology modeling of CGRP antibody Fv fragmentswas carried out. CGRP sequences were BLAST searched against PDB_Antibodydatabase for identifying the best templates for Fv fragments andespecially for building the domain interface. Structural template 113G(Crystal structure of an ampicillin single chain fv, form 1) wasselected, identity=75%. Altogether, there were 17 amino acids, 9 fromHCVR (K12V, V20I, R38K, M48I, V68A, M70L, R72V, R98S and V115L) and 8from LCVR (L4M, A13T, I21V, Q37R, A43S, L46A, L78V and Y87I) identifiedfor replacement. Back mutated antibodies were then expressed in HEK293cells and assessed. Based on the assessment, the humanized heavy chainsconstructed for use in screening for lead humanized antibodies werenamed as H1, H2, H3, and H4 and comprise the sequences set forth in SEQID NOs: 41, 43, 45 and 47, respectively, while the resultant humanizedlight chains were named as L1, L2, L3 and L4 and comprise the sequencesset forth in SEQ ID NOs: 49, 51, 53 and 55, respectively.

Using various combinations of H1-H4 and L1-L4, sixteen humanizedantibodies were expressed in HEK 293-6E cells. Briefly, nucleic acidsencoded by any one of SEQ ID NOs: 42 (H1), 44 (H2), 46 (H3), or 48 (H4)and any one of SEQ ID NOs: 50 (L1), 52 (L2), 54 (L3) or 56 (L4), each ofwhich includes a leader sequence, were amplified and inserted into pTT5to make an expression plasmid of the full-length IgG. The heavy chainand light chain expression plasmids were used to co-transfect 100 mLHEK293-6E cells. The recombinant IgG secreted into to media was purifiedusing protein A affinity. The purified antibody was buffer-exchangedinto PBS using PD-10 desalting column. The purified IgG migrated as ˜170kDa band in SDS-PAGE under non-reducing conditions, and the yield formthe 100 mL culture was more than 20 mg/L. The HC and LC amino acidsequences for the 16 humanized CGRP antibodies are summarized in Table5:

TABLE 5 Humanized IgG HC LC 1 (H1/L1) SEQ ID NO: 41 SEQ ID NO: 49 2(H1/L2) SEQ ID NO: 41 SEQ ID NO: 51 3 (H1/L3) SEQ ID NO: 41 SEQ ID NO:53 4 (H1/L4) SEQ ID NO: 41 SEQ ID NO: 55 5 (H2/L1) SEQ ID NO: 43 SEQ IDNO: 49 6 (H2/L2) SEQ ID NO: 43 SEQ ID NO: 51 7 (H2/L3) SEQ ID NO: 43 SEQID NO: 53 8 (H2/L4) SEQ ID NO: 43 SEQ ID NO: 55 9 (H3/L1) SEQ ID NO: 45SEQ ID NO: 49 10 (H3/L2)  SEQ ID NO: 45 SEQ ID NO: 51 11 (H3/L3)  SEQ IDNO: 45 SEQ ID NO: 53 12 (H3/L4)  SEQ ID NO: 45 SEQ ID NO: 55 13 (H4/L1) SEQ ID NO: 47 SEQ ID NO: 49 14 (H4/L2)  SEQ ID NO: 47 SEQ ID NO: 51 15(H4/L3)  SEQ ID NO: 47 SEQ ID NO: 53 16 (H4/L4)  SEQ ID NO: 47 SEQ IDNO: 55

Affinity ranking of the sixteen humanized IgGs was performed usingBiacore T200 (GE Healthcare). Anti-human Fc gamma specific antibody wasimmobilized onto the sensor chip using amine coupling method. Sixteenhumanized antibodies secreted to the culture medium plus the parentalantibody were injected and captured by anti-human Fc antibody via Fc(capture phase) individually. After equilibration, Ag CGRP was injectedfor 300 seconds (association phase) followed by the injection of runningbuffer for 900s (dissociation phase). Responses of reference flow cell(flow cell 1) were subtracted from those of humanized antibodies flowcells during each cycle. The surface was regenerated before theinjection of other humanized antibodies. The process was repeated untilall antibodies are analyzed. The off-rates of humanized antibodies wereobtained from fitting the experimental data locally to 1:1 interactionmodel using the Biacore T200 evaluation software. The antibodies wereranked by their dissociation rate constants (off-rates, k_(d)). Thebinders that interact with Ag CGRP with similar affinity to parentalantibody were selected.

Based on the affinity rankings, four IgGs: 1) IgG 10 (H3/L2)(alsoreferred to hereinafter as “REMD 128”); 2) IgG 11 (H3/L3)(also referredto hereinafter as “REMD 128.1”); 3) IgG 15 (H4/L3)(also referred tohereinafter as “REMD 128.2”) and 4) IgG 14 (H4/L2)(also referred tohereinafter as “REMD 128.3”) were selected for expression in HEK293 cellculture. The recombinant IgGs secreted to the medium and were purifiedusing protein A affinity chromatography. Evaluating from the SDS-PAGE,the purity of humanized IgGs were all over 90%. The affinities ofpurified antibodies binding to CGRP were determined using a SurfacePlasmon Resonance (SPR) biosensor, Biacore 8k. Antibodies wereimmobilized on the sensor chip through amine coupling method. AntigenCGRP was used as the analyte. The data of dissociation (kd) andassociation (ka) rate constants were obtained using Biacore 8kevaluation software. The equilibrium dissociation constants (KD) werecalculated from the ratio of kd over ka. The results are summarized inTable 7:

TABLE 7 k_(a) Rmax Chi² Ligand Analyte (1/Ms) k_(d) (1/s) K_(D) (M) (RU)(RU²) Chimeric CGRP 3.37E+06 1.19E−05 3.53E−12 33.3 0.0184 IgG4 REMDCGRP 4.23E+06 5.79E−06 1.37E−12 33.7 0.0286 128 REMD CGRP 4.86E+067.61E−06 1.57E−12 43   0.0317 128.1 REMD CGRP 4.97E+06 7.57E−06 1.52E−1237.5 0.0254 128.2 REMD CGRP 4.55E+06 7.01E−06 1.54E−12 33.5 0.0245 128.3As depicted in Table 7, the 4 humanized antibodies retain comparableantigen-binding affinities to the chimeric antibody.

The four humanized antibodies were then evaluated in the SK-N-MC cellbased assay described in Example 1. The results are summarized in FIG. 3and Table 8:

TABLE 8 Ligand SK-N-MC IC50 (pM) Murine 30B8G9B11 152 Chimeric IgG4 152REMD 128 49.2 REMD 128.1 38.9 REMD 128.2 60.4 REMD 128.3 59.8As depicted in Table 7, the 4 humanized antibodies retain comparableantigen-binding affinities to the chimeric antibody and inhibited theproduction of cAMP with 1050 between 49-60 pM Ab as required for aclinical mAb therapeutic.

All of the articles and methods disclosed and claimed herein can be madeand executed without undue experimentation in light of the presentdisclosure. While the articles and methods of this invention have beendescribed in terms of preferred embodiments, it will be apparent tothose of skill in the art that variations may be applied to the articlesand methods without departing from the spirit and scope of theinvention. All such variations and equivalents apparent to those skilledin the art, whether now existing or later developed, are deemed to bewithin the spirit and scope of the invention as defined by the appendedclaims. All patents, patent applications, and publications mentioned inthe specification are indicative of the levels of those of ordinaryskill in the art to which the invention pertains. All patents, patentapplications, and publications are herein incorporated by reference intheir entirety for all purposes and to the same extent as if eachindividual publication was specifically and individually indicated to beincorporated by reference in its entirety for any and all purposes. Theinvention illustratively described herein suitably may be practiced inthe absence of any element(s) not specifically disclosed herein. Thus,it should be understood that although the present invention has beenspecifically disclosed by preferred embodiments and optional features,modification and variation of the concepts herein disclosed may beresorted to by those skilled in the art, and that such modifications andvariations are considered to be within the scope of this invention asdefined by the appended claims.

Sequence Listings

The nucleic and amino acid sequences listed in the accompanying sequencelisting are shown using standard letter abbreviations for nucleotidebases and three letter code for amino acids, as defined in 37 C.F.R.1.822.

SEQ ID NO: 1 is an amino acid sequence comprising a human CGRP.

SEQ ID NO: 2 is the amino acid sequence of a heavy chain CDR1 in amonoclonal antibody which specifically binds CGRP.

SEQ ID NOs: 3-5 are the amino acid sequences of a heavy chain CDR2 in amonoclonal antibody which specifically binds CGRP.

SEQ ID NOs: 6-8 are the amino acid sequences of a heavy chain CDR3 in amonoclonal antibody which specifically binds CGRP.

SEQ ID NOs: 9-11 are the amino acid sequences of a light chain CDR1 in amonoclonal antibody which specifically binds CGRP.

SEQ ID NO: 12-13 is the amino acid sequences of a light chain CDR2 in amonoclonal antibody which specifically binds CGRP.

SEQ ID NOs: 14-16 are the amino acid sequences of a light chain CDR3 ina monoclonal antibody which specifically binds CGRP.

SEQ ID NOs: 17, 19 and 21 are amino acid sequences of a heavy chainvariable region of murine monoclonal antibodies which specifically bindCGRP.

SEQ ID NOs: 18, 20 and 22 are nucleic acid sequences encoding a heavychain variable region of murine monoclonal antibodies which specificallybind CGRP.

SEQ ID NOs: 23, 25 and 27 are amino acid sequences of a light chainvariable region of murine monoclonal antibodies which specifically bindCGRP.

SEQ ID NOs: 24, 26 and 28 are nucleic acid sequences encoding a lightchain variable region of murine monoclonal antibodies which specificallybind CGRP.

SEQ ID NO: 29 is the amino acid sequence of a heavy chain of amurine-human chimeric antibody which specifically binds CGRP.

SEQ ID NO: 30 is the nucleic acid sequence of a heavy chain of amurine-human chimeric antibody which specifically binds CGRP.

SEQ ID NO: 31 is the amino acid sequence of a light chain of amurine-human chimeric antibody which specifically binds CGRP.

SEQ ID NO: 32 is the nucleic acid sequence of a light chain of amurine-human chimeric antibody which specifically binds CGRP.

SEQ ID NOs: 33, 35, 37 and 39 are amino acid sequences of a heavy chainvariable region of humanized monoclonal antibodies which specificallybinds CGRP.

SEQ ID NOs: 34, 36, 38 and 40 are amino acid sequences of a light chainvariable regions of humanized monoclonal antibodies which specificallybinds CGRP.

SEQ ID NOs: 41, 43, 45 and 47 are the amino acid sequences of a heavychain of humanized monoclonal antibodies which specifically binds CGRP.

SEQ ID NOs: 42, 44, 46 and 48 are nucleic acid sequences of a heavychain of humanized monoclonal antibodies which specifically binds CGRP.

SEQ ID NOs: 49, 51, 53 and 55 are amino acid sequences of a light chainof humanized monoclonal antibodies which specifically binds CGRP.

SEQ ID NOs: 50, 52, 54 and 56 are nucleic acid sequences of a lightchain of humanized monoclonal antibodies which specifically binds CGRP.

SEQ ID NOs: 57 and 58 are the amino acid sequences of a light chainconstant region of a monoclonal antibody which specifically binds CGRP.

SEQ ID NO: 59 is the amino acid sequence of a heavy chain constantregion of a monoclonal antibody which specifically binds CGRP.

SEQ ID NO: 60 is an amino acid sequence of a heavy chain variable regionof humanized monoclonal antibody which specifically binds CGRP.

SEQ ID NO: 61 is an amino acid sequence of a light chain variable regionof humanized monoclonal antibody which specifically binds CGRP.

SEQUENCE LISTINGS SEQ ID NO: 1-CGRP amino acid sequenceACDTATCVTHRLAGLLSRSGGVVKNNFVPTNVGSKAFSEQ ID NO: 2-Murine monoclonal antibody heavy chain CDR1 amino acid sequenceDYYMNSEQ ID NO: 3-Murine monoclonal antibody heavy chain CDR2 amino acid sequenceDIN PNNGGTTYNQKFKGSEQ ID NO: 4-Murine monoclonal antibody heavy chain CDR2 amino acid sequenceDVNPNNGDTTYNQKFKGSEQ ID NO: 5-Murine monoclonal antibody heavy chain CDR2 amino acid sequenceDVNPNNGGTHYNQKFKGSEQ ID NO: 6-Murine monoclonal antibody heavy chain CDR3 amino acid sequenceITIVPYYFDYSEQ ID NO: 7-Murine monoclonal antibody heavy chain CDR3 amino acid sequenceITIVPLYFDFSEQ ID NO: 8-Murine monoclonal antibody heavy chain CDR3 amino acid sequenceITIVPVYFDFSEQ ID NO: 9-Murine monoclonal antibody light chain CDR1 amino acid sequenceKASQNVGSNVASEQ ID NO: 10-Murine monoclonal antibody light chain CDR1 amino acid sequenceKASQNVGINVASEQ ID NO: 11-Murine monoclonal antibody light chain CDR1 amino acid sequenceKASQNVGPNVASEQ ID NO: 12-Murine monoclonal antibody light chain CDR2 amino acid sequenceSASFRYSSEQ ID NO: 13-Murine monoclonal antibody light chain CDR2 amino acid sequenceSASYRYSSEQ ID NO: 14-Murine monoclonal antibody light chain CDR3 amino acid sequenceQQYNSYPYTSEQ ID NO: 15- Murine monoclonal antibody light chain CDR3 amino acid sequenceQQYNTYPYTSEQ ID NO: 16- Murine monoclonal antibody light chain CDR3 amino acid sequenceQQYNYYPYTSEQ ID NO: 17? Murine monoclonal antibody heavy chain variable region amino acidsequence EVQLQQSGPELVKPGASVKISCKASGYTFTDYYMNWVKQSHGKSLEWIGDINPNNGGTTYNQKFKGKATLTVDKSSTTAYMELRSLTSEDSAVYYCAVITIVPYYFDYWGQGTTLTVSSSEQ ID NO: 18-Murine monoclonal antibody heavy chain variable region nucleic acidsequencegaggtccagctgcaacaatctggacctgagctggtgaagcctggggcttcagtgaagatatcctgtaaggcttctggatacacgttcactgactactacatgaactgggtgaagcagagccatggaaagagccttgagtggattggagatattaatcctaacaatggtggtactacttacaaccagaagttcaagggcaaggccacattgactgtagacaagtcctccaccacagcctacatggagctccgcagcctgacatctgaggactctgcagtctattactgtgcagtcattactatagtcccctactactttgactactggggccaaggcaccactctcacagtctcctcaSEQ ID NO: 19-Murine monoclonal antibody heavy chain variable region amino acidsequence EVQLLQSGPELMKPGTSVKISCKASGYTFTDYYMNWVKQSHGKSLEWIGDVNPNNGDTTYNQKFKGKATLTIDKSSSTAYMELRSLTSEDSAVYYCASITIVPLYFDFWGQGTTLAVSSSEQ ID NO: 20-Murine monoclonal antibody heavy chain variable region nucleic acidsequencegaggtccagctgttacaatctggacctgagctgatgaagcctgggacttcagtgaagatatcctgtaaggcttctggatacacgttcactgactactacatgaactgggtgaagcagagccatggtaagagccttgagtggattggagatgttaatcctaacaatggtgatactacctacaaccagaagttcaagggcaaggccacattgactatagacaagtcctccagcacagcctacatggaactccgcagcctgacatctgaggactctgcagtctactactgtgcaagtattacgattgttcctctttactttgacttctggggccaaggcaccactctcgcagtctcctcaSEQ ID NO: 21-Murine monoclonal antibody heavy chain variable region amino acidsequence EVQLQQSGPELVKPGASVKISCKASGYTFTDYYMNWVKQSHGKSLEWIGDVNPNNGGTHYNQKFKGKATLTVDKSSSTAYMELRSLTSEDSAVYYCASIIIVPVYFDFWGQGTTLTVSSSEQ ID NO: 22-Murine monoclonal antibody heavy chain variable region nucleic acidsequencegaggtccagctgcaacaatctggacctgaactggtgaagcctggggcttcagtgaagatatcctgtaaggcttctggatacacgttcactgactactacatgaactgggtgaagcagagccatggaaagagccttgagtggattggagatgttaatcctaacaatggtggtactcactacaaccagaagttcaagggcaaggccacattgactgtagacaagtcctccagtacagcctacatggagctccgcagcctgacatctgaggactctgcagtctattactgtgcaagtattacgattgtacctgtttactttgacttctggggccaaggcaccactctcacagtctcctcaSEQ ID NO: 23-Murine monoclonal antibody light chain variable region amino acid sequenceDIVMTQSQKFMSTSVGDRVSVTCKASQNVGSNVAWYQQKPGQSPKALIYSASFRYSRVPDRFSGSGSGTDFTLTISNVQSEDLADYFCQQYNSYPYTFGAGTKLELKSEQ ID NO: 24-Murine monoclonal antibody light chain variable region nucleic acid sequencegacattgttatgacccagtctcaaaaattcatgtccacatcagtaggagacagggtcagcgtcacctgcaaggccagtcagaatgtgggtagtaatgtagcctggtatcaacagaaaccagggcaatctcctaaagcactgatttactcggcatcctttcgttacagtagagtccctgatcgcttctcaggcagtggatctgggacagatttcactctcaccatcagcaatgtgcagtctgaagacttggcagactatttctgtcagcaatataacagttatccttacacgttcggtgctgggaccaagctggagctgaaaSEQ ID NO: 25-Murine monoclonal antibody light chain variable region amino acid sequenceDIVMTQSQKFMSTSVGDRVSVTCKASQNVGINVAWYQQKPGQSPKALLYSASYRYSGVPDRFTGSGSGTDFTLTISNVKSGDLAEYFCQQYNTYPYTFGGGTKLEIKSEQ ID NO: 26-Murine monoclonal antibody light chain variable region nucleic acid sequencegacattgtgatgacccagtctcaaaaattcatgtccacatcagtgggagacagggtcagcgtcacctgcaaggccagtcagaatgtgggtattaatgtagcctggtatcaacagaagccaggacaatctcctaaagcactgctttactcggcatcctaccgatatagtggagtccctgatcgcttcacaggcagtggttctgggacagatttcactctcaccatcagcaatgtgaagtctggagacttggcagagtatttctgtcagcaatataacacctatccgtacacgttcggaggggggaccaagctggaaataaaaSEQ ID NO: 27-Murine monoclonal antibody light chain variable region amino acid sequenceDIVMTQSQKFMSTSVGDRVSVTCKASQNVGPNVAWYRQKPGQSPKALIYSASYRYSGVPDRFTGSGSGTDFTLTISNVQSEDLADYICQQYNYYPYTFGGGTKLEIKSEQ ID NO: 28-Murine monoclonal antibody light chain variable region nucleic acid sequencegacattgtgatgacccagtctcaaaaattcatgtccacatcagtaggagacagggtcagcgtcacctgcaaggccagtcagaatgtgggtcctaatgtagcctggtatcgacagaaaccaggacaatctcctaaagctctgatttactcggcatcctaccggtacagtggagtccctgatcgcttcacaggcagtggatctgggacagatttcactctcaccatcagcaatgtgcaatctgaagacttggcagactatatctgtcagcagtataactactatccgtacacgttcggaggggggaccaaactggaaataaaaSEQ ID NO: 29-Heavy chain amino acid sequence of amurine-human chimeric antibodyMGWSWILLFLLSVTAGVHSEVQLQQSGPELVKPGASVKISCKASGYTFTDYYMNWVKQSHGKSLEWIGDVNPNNGGTHYNQKFKGKATLTVDKSSSTAYMELRSLTSEDSAVYYCASITIVPVYFDFWGQGTTLTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGKSEQ ID NO: 30-Heavy chain nucleic acid sequence of amurine-human chimeric antibodyatgggctggagctggatcctgctgttcctcctgagcgtgacagcaggagtgcacagcgaggtccagctgcaacaatctggacctgaactggtgaagcctggggcttcagtgaagatatcctgtaaggcttctggatacacgttcactgactactacatgaactgggtgaagcagagccatggaaagagccttgagtggattggagatgttaatcctaacaatggtggtactcactacaaccagaagttcaagggcaaggccacattgactgtagacaagtcctccagtacagcctacatggagctccgcagcctgacatctgaggactctgcagtctattactgtgcaagtattacgattgtacctgtttactttgacttctggggccaaggcaccactctcacagtctcctcagcctctacaaagggcccctccgtgtttccactggctccctgcagcaggtctacatccgagagcaccgctgctctgggatgtctggtgaaggattacttccctgagccagtgaccgtgagctggaactccggagctctgacatccggagtgcacacctttcctgctgtgctgcagagctctggcctgtacagcctgtccagcgtggtgacagtgccatcttccagcctgggcaccaagacatatacctgcaacgtggaccataagcccagcaataccaaggtggataagagagtggagtctaagtacggaccaccttgcccaccatgtccagctcctgagtttctgggaggaccatccgtgttcctgtttcctccaaagcctaaggacaccctgatgatctctcgcacacccgaggtgacctgtgtggtggtggacgtgtcccaggaggatcctgaggtgcagttcaactggtacgtggatggcgtggaggtgcacaatgctaagaccaagcctagggaggagcagtttaacagcacataccgggtggtgtctgtgctgaccgtgctgcatcaggactggctgaacggcaaggagtataagtgcaaggtgagcaataagggcctgccatcttccatcgagaagacaatctctaaggctaagggacagcctagggagccacaggtgtacaccctgcccccttcccaggaggagatgacaaagaaccaggtgagcctgacctgtctggtgaagggcttctatccttctgacatcgctgtggagtgggagtccaatggccagccagagaacaattacaagaccacaccacccgtgctggactccgatggcagcttctttctgtattccaggctgaccgtggataagagccggtggcaggagggcaatgtgttttcttgttccgtgatgcacgaagcactgcacaaccactacactcagaagtccctgtcactgtccctgggcaagSEQ ID NO: 31-Light chain amino acid sequence of amurine-human chimeric antibodyMGWSWILLFLLSVTAGVHSDIVMTQSQKFMSTSVGDRVSVTCKASQNVGPNVAWYRQKPGQSPKALIYSASYRYSGVPDRFTGSGSGTDFTLTISNVQSEDLADYICQQYNYYPYTFGGGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGECSEQ ID NO: 32-Light chain nucleic acid sequence of amurine-human chimeric antibodyatgggctggagctggatcctgctgttcctcctgagcgtgacagcaggagtgcacagcgaggtccagctgttacaatctggacctgagctgatgaagcctgggacttcagtgaagatatcctgtaaggcttctggatacacgttcactgactactacatgaactgggtgaagcagagccatggtaagagccttgagtggattggagatgttaatcctaacaatggtgatactacctacaaccagaagttcaagggcaaggccacattgactatagacaagtcctccagcacagcctacatggaactccgcagcctgacatctgaggactctgcagtctactactgtgcaagtattacgattgttcctctttactttgacttctggggccaaggcaccactctcgcagtctcctcacgaacggtggctgcaccatctgtcttcatcttcccgccatctgatgagcagttgaaatctggaactgcctctgttgtgtgcctgctgaataacttctatcccagagaggccaaagtacagtggaaggtggataacgccctccaatcgggtaactcccaggagagtgtcacagagcaggacagcaaggacagcacctacagcctcagcagcaccctgacgctgagcaaagcagactacgagaaacacaaagtctacgcctgcgaagtcacccatcagggcctgagctcgcccgtcacaaagagcttcaacaggggagagtgtSEQ ID NO: 33-Humanized heavy chain variable region amino acid sequenceQVQLVQSGAEVKKPGASVKVSCKASGYTFTDYYMNWVRQAPGQGLEWMGDVNPNNGGTHYNQKFKGRVTMTRDTSISTAYMELSRLRSDDTAVYYCARITIVPVYFDFWGQGTLVTVSSSEQ ID NO: 34-Humanized light chain variable region amino acid sequenceDIQLTQSPSFLSASVGDRVTITCKASQNVGPNVAWYQQKPGKAPKLLIYSASYRYSGVPSRFSGSGSGTEFTLTISSLQPEDFATYYCQQYNYYPYTFGQGTKLEIKSEQ ID NO: 35-Humanized heavy chain variable region amino acid sequenceQVQLVQSGAEVVKPGASVKVSCKASGYTFTDYYMNWVRQAPGQGLEWMGDVNPNNGGTHYNQKFKGRVTMTVDTSISTAYMELSRLRSDDTAVYYCASITIVPVYFDFWGQGTLVTVSSSEQ ID NO: 36-Humanized light chain variable region amino acid sequenceDIQLTQSPSFLSASVGDRVTITCKASQNVGPNVAWYRQKPGKAPKALIYSASYRYSGVPSRFSGSGSGTEFTLTISSLQPEDFATYICQQYNYYPYTFGQGTKLEIKSEQ ID NO: 37-Humanized heavy chain variable region amino acid sequenceQVQLVQSGAEVVKPGASVKVSCKASGYTFTDYYMNWVKQAPGQGLEWIGDVNPNNGGTHYNQKFKGRVTLTVDTSISTAYMELSRLRSDDTAVYYCASIIIVPVYFDFWGQGTLVTVSSSEQ ID NO: 38-Humanized light chain variable region amino acid sequenceDIQLTQSPSFLSTSVGDRVTITCKASQNVGPNVAWYRQKPGKSPKALIYSASYRYSGVPSRFSGSGSGTEFTLTISSLQPEDFATYICQQYNYYPYTFGQGTKLEIKSEQ ID NO: 39-Humanized heavy chain variable region amino acid sequenceQVQLVQSGAEVVKPGASVKISCKASGYTFTDYYMNWVKQAPGQGLEWIGDVNPNNGGTHYNQKFKGRATLTVDTSISTAYMELSRLRSDDTAVYYCASITIVPVYFDFWGQGTLLTVSSSEQ ID NO: 40-Humanized light chain variable region amino acid sequenceDIQMTQSPSFLSTSVGDRVTVTCKASQNVGPNVAWYRQKPGKSPKALIYSASYRYSGVPSRFSGSGSGTEFTLTISSVQPEDFATYICQQYNYYPYTFGQGTKLEIKSEQ ID NO: 41-Humanized heavy chain amino acid sequenceMGWSWILLFLLSVTAGVHSQVQLVQSGAEVKKPGASVKVSCKASGYTFTDYYMNWVRQAPGQGLEWMGDVNPNNGGTHYNQKFKGRVTMTRDTSISTAYMELSRLRSDDTAVYYCARITIVPVYFDFWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGKSEQ ID NO: 42-Humanized heavy chain nucleic acid sequenceatg ggctggagctggatcctgctgttcctcctgagcgtgacagcaggagtgcacagccaggtccagctggtccagtcaggggcagaagtgaagaaacccggagcaagtgtgaaggtgtcatgcaaagcctcaggatatacattcacagactactatatgaactgggtgcgacaggcaccaggacagggactggagtggatgggggatgtcaaccctaacaatggcgggactcactacaatcagaagttcaaaggcagggtgaccatgacacgcgacactagcatctccaccgcctatatggaactgtctcggctgagaagcgacgataccgccgtctactattgcgctagaattactattgtgcccgtgtattttgatttttggggacagggaactctggtcaccgtctcatccgcctctacaaagggcccctccgtgtttccactggctccctgcagcaggtctacatccgagagcaccgctgctctgggatgtctggtgaaggattacttccctgagccagtgaccgtgagctggaactccggagctctgacatccggagtgcacacctttcctgctgtgctgcagagctctggcctgtacagcctgtccagcgtggtgacagtgccatcttccagcctgggcaccaagacatatacctgcaacgtggaccataagcccagcaataccaaggtggataagagagtggagtctaagtacggaccaccttgcccaccatgtccagctcctgagtttctgggaggaccatccgtgttcctgtttcctccaaagcctaaggacaccctgatgatctctcgcacacccgaggtgacctgtgtggtggtggacgtgtcccaggaggatcctgaggtgcagttcaactggtacgtggatggcgtggaggtgcacaatgctaagaccaagcctagggaggagcagtttaacagcacataccgggtggtgtctgtgctgaccgtgctgcatcaggactggctgaacggcaaggagtataagtgcaaggtgagcaataagggcctgccatcttccatcgagaagacaatctctaaggctaagggacagcctagggagccacaggtgtacaccctgcccccttcccaggaggagatgacaaagaaccaggtgagcctgacctgtctggtgaagggcttctatccttctgacatcgctgtggagtgggagtccaatggccagccagagaacaattacaagaccacaccacccgtgctggactccgatggcagcttctttctgtattccaggctgaccgtggataagagccggtggcaggagggcaatgtgttttcttgttccgtgatgcacgaagcactgcacaaccactacactcagaagtccctgtcactgtccctgggcaagSEQ ID NO: 43-Humanized heavy chain amino acid sequenceMGWSWILLFLLSVTAGVHSQVQLVQSGAEVVKPGASVKVSCKASGYTFTDYYMNWVRQAPGQGLEWMGDVNPNNGGTHYNQKFKGRVTMTVDTSISTAYMELSRLRSDDTAVYYCASITIVPVYFDFWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNV FSCSVMHEALHNHYTQKSLSLSLGKSEQ ID NO: 44-Humanized heavy chain nucleic acid sequenceatgggctggagctggatcctgctgttcctcctgagcgtgacagcaggagtgcacagccaggtccagctggtccagtcaggagcagaggtcgtgaaacccggagcaagcgtcaaggtctcatgcaaagcaagcggctatacattcacagactactatatgaactgggtgaggcaggcaccaggacagggactggagtggatgggggatgtcaaccctaacaatggcgggactcactacaatcagaagttcaaaggccgggtgaccatgacagtcgacactagcatctccaccgcctatatggaactgtctcggctgagaagtgacgataccgccgtgtactattgcgcttccattactatcgtgcccgtctactttgacttctggggacaggggacactggtgaccgtctcatccgcctctacaaagggcccctccgtgtttccactggctccctgcagcaggtctacatccgagagcaccgctgctctgggatgtctggtgaaggattacttccctgagccagtgaccgtgagctggaactccggagctctgacatccggagtgcacacctttcctgctgtgctgcagagctctggcctgtacagcctgtccagcgtggtgacagtgccatcttccagcctgggcaccaagacatatacctgcaacgtggaccataagcccagcaataccaaggtggataagagagtggagtctaagtacggaccaccttgcccaccatgtccagctcctgagtttctgggaggaccatccgtgttcctgtttcctccaaagcctaaggacaccctgatgatctctcgcacacccgaggtgacctgtgtggtggtggacgtgtcccaggaggatcctgaggtgcagttcaactggtacgtggatggcgtggaggtgcacaatgctaagaccaagcctagggaggagcagtttaacagcacataccgggtggtgtctgtgctgaccgtgctgcatcaggactggctgaacggcaaggagtataagtgcaaggtgagcaataagggcctgccatcttccatcgagaagacaatctctaaggctaagggacagcctagggagccacaggtgtacaccctgcccccttcccaggaggagatgacaaagaaccaggtgagcctgacctgtctggtgaagggcttctatccttctgacatcgctgtggagtgggagtccaatggccagccagagaacaattacaagaccacaccacccgtgctggactccgatggcagcttctttctgtattccaggctgaccgtggataagagccggtggcaggagggcaatgtgttttcttgttccgtgatgcacgaagcactgcacaaccactacactcagaagtccctgtcactgtccctgggcaagSEQ ID NO: 45-Humanized heavy chain amino acid sequenceMGWSWILLFLLSVTAGVHSQVQLVQSGAEVVKPGASVKVSCKASGYTFTDYYMNWVKQAPGQGLEWIGDVNPNNGGTHYNQKFKGRVTLTVDTSISTAYMELSRLRSDDTAVYYCASIIIVPVYFDFWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGKSEQ ID NO: 46-Humanized heavy chain nucleic acid sequenceatgggctggagctggatcctgctgttcctcctgagcgtgacagcaggagtgcacagccaggtccagctggtccagtcaggggcagaggtggtcaaacccggagcaagcgtgaaagtctcatgcaaagcaagcggctatacattcacagactactatatgaactgggtgaagcaggcaccaggacagggactggagtggatcggggatgtcaaccctaacaatggcgggactcactacaatcagaagttcaaaggcagggtgaccctgacagtcgacactagcatctccaccgcctatatggaactgtctcggctgagaagtgacgataccgccgtgtactattgcgcttccattactattgtgcccgtctattttgacttctggggacaggggacactggtgaccgtctcctcagcctctacaaagggcccctccgtgtttccactggctccctgcagcaggtctacatccgagagcaccgctgctctgggatgtctggtgaaggattacttccctgagccagtgaccgtgagctggaactccggagctctgacatccggagtgcacacctttcctgctgtgctgcagagctctggcctgtacagcctgtccagcgtggtgacagtgccatcttccagcctgggcaccaagacatatacctgcaacgtggaccataagcccagcaataccaaggtggataagagagtggagtctaagtacggaccaccttgcccaccatgtccagctcctgagtttctgggaggaccatccgtgttcctgtttcctccaaagcctaaggacaccctgatgatctctcgcacacccgaggtgacctgtgtggtggtggacgtgtcccaggaggatcctgaggtgcagttcaactggtacgtggatggcgtggaggtgcacaatgctaagaccaagcctagggaggagcagtttaacagcacataccgggtggtgtctgtgctgaccgtgctgcatcaggactggctgaacggcaaggagtataagtgcaaggtgagcaataagggcctgccatcttccatcgagaagacaatctctaaggctaagggacagcctagggagccacaggtgtacaccctgcccccttcccaggaggagatgacaaagaaccaggtgagcctgacctgtctggtgaagggcttctatccttctgacatcgctgtggagtgggagtccaatggccagccagagaacaattacaagaccacaccacccgtgctggactccgatggcagcttctttctgtattccaggctgaccgtggataagagccggtggcaggagggcaatgtgttttcttgttccgtgatgcacgaagcactgcacaaccactacactcagaagtccctgtcactgtccctgggcaagSEQ ID NO: 47-Humanized heavy chain amino acid sequenceMGWSWILLFLLSVTAGVHSQVQLVQSGAEVVKPGASVKISCKASGYTFTDYYMNWVKQAPGQGLEWIGDVNPNNGGTHYNQKFKGRATLTVDTSISTAYMELSRLRSDDTAVYYCASITIVPVYFDFWGQGTLLTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGKSEQ ID NO: 48-Humanized heavy chain nucleic acid sequenceatgggctggagctggatcctgctgttcctcctgagcgtgacagcaggagtgcacagccaggtgcagctggtccagtcaggggcagaggtcgtcaaacccggagcaagcgtgaagattagttgtaaggcatcaggctatactttcacagactactatatgaactgggtgaagcaggctccaggacagggactggagtggatcggggatgtcaaccctaacaatggcgggactcactacaatcagaagttcaaaggcagggcaaccctgacagtggacactagcatctccaccgcctatatggaactgtctcggctgagaagtgacgataccgccgtctactattgcgcttccattaccattgtgccagtctattttgatttttggggacagggaactctgctgacagtctcctcagcctctacaaagggcccctccgtgtttccactggctccctgcagcaggtctacatccgagagcaccgctgctctgggatgtctggtgaaggattacttccctgagccagtgaccgtgagctggaactccggagctctgacatccggagtgcacacctttcctgctgtgctgcagagctctggcctgtacagcctgtccagcgtggtgacagtgccatcttccagcctgggcaccaagacatatacctgcaacgtggaccataagcccagcaataccaaggtggataagagagtggagtctaagtacggaccaccttgcccaccatgtccagctcctgagtttctgggaggaccatccgtgttcctgtttcctccaaagcctaaggacaccctgatgatctctcgcacacccgaggtgacctgtgtggtggtggacgtgtcccaggaggatcctgaggtgcagttcaactggtacgtggatggcgtggaggtgcacaatgctaagaccaagcctagggaggagcagtttaacagcacataccgggtggtgtctgtgctgaccgtgctgcatcaggactggctgaacggcaaggagtataagtgcaaggtgagcaataagggcctgccatcttccatcgagaagacaatctctaaggctaagggacagcctagggagccacaggtgtacaccctgcccccttcccaggaggagatgacaaagaaccaggtgagcctgacctgtctggtgaagggcttctatccttctgacatcgctgtggagtgggagtccaatggccagccagagaacaattacaagaccacaccacccgtgctggactccgatggcagcttctttctgtattccaggctgaccgtggataagagccggtggcaggagggcaatgtgttttcttgttccgtgatgcacgaagcactgcacaaccactacactcagaagtccctgtcactgtccctgggcaagSEQ ID NO: 49-Humanized light chain amino acid sequenceMGWSWILLFLLSVTAGVHSDIQLTQSPSFLSASVGDRVTITCKASQNVGPNVAWYQQKPGKAPKLLIYSASYRYSGVPSRFSGSGSGTEFTLTISSLQPEDFATYYCQQYNYYPYTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGECSEQ ID NO: 50-Humanized light chain nucleic acid sequenceatgggctggagctggatcctgctgttcctcctgagcgtgacagcaggagtgcacagcgacatccagctgacccagtccccctcattcctgtccgcaagtgtgggcgaccgagtcaccatcacctgtaaggcaagccagaacgtgggccccaacgtggcatggtaccagcagaagcccgggaaagcccctaagctgctgatctattctgctagttaccggtattctggcgtcccaagcagattctcaggcagcgggtccggaactgagtttaccctgacaattagctccctgcagcccgaagacttcgccacctactattgtcagcagtacaactattacccatacaccttcgggcagggcactaaactggaaatcaaacgaacggtggctgcaccatctgtcttcatcttcccgccatctgatgagcagttgaaatctggaactgcctctgttgtgtgcctgctgaataacttctatcccagagaggccaaagtacagtggaaggtggataacgccctccaatcgggtaactcccaggagagtgtcacagagcaggacagcaaggacagcacctacagcctcagcagcaccctgacgctgagcaaagcagactacgagaaacacaaagtctacgcctgcgaagtcacccatcagggcctgagctcgcccgtcacaaagagcttcaacaggggagagtgt SEQ ID NO: 51-Humanized light chain amino acid sequenceMGWSWILLFLLSVTAGVHSDIQLTQSPSFLSASVGDRVTITCKASQNVGPNVAWYRQKPGKAPKALIYSASYRYSGVPSRFSGSGSGTEFTLTISSLQPEDFATYICQQYNYYPYTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGECSEQ ID NO: 52-Humanized light chain nucleic acid sequenceatgggctggagctggatcctgctgttcctcctgagcgtgacagcaggagtgcacagcgacatccagctgacccagtccccatccttcctgagcgcaagcgtcggagacagagtgaccattacctgcaaagcatcccagaacgtgggccccaacgtggcatggtacaggcagaagcccgggaaagcccctaaggctctgatctattctgccagttaccggtattctggcgtcccaagcagattctcaggcagcgggtccggaactgagtttaccctgacaatcagctccctgcagcccgaagacttcgctacctacatttgtcagcagtacaactattatccttacacctttgggcagggcactaaactggaaatcaagcgaacggtggctgcaccatctgtcttcatcttcccgccatctgatgagcagttgaaatctggaactgcctctgttgtgtgcctgctgaataacttctatcccagagaggccaaagtacagtggaaggtggataacgccctccaatcgggtaactcccaggagagtgtcacagagcaggacagcaaggacagcacctacagcctcagcagcaccctgacgctgagcaaagcagactacgagaaacacaaagtctacgcctgcgaagtcacccatcagggcctgagctcgcccgtcacaaagagcttcaacaggggagagtgt SEQ ID NO: 53-Humanized light chain amino acid sequenceMGWSWILLFLLSVTAGVHSDIQLTQSPSFLSTSVGDRVTITCKASQNVGPNVAWYRQKPGKSPKALIYSASYRYSGVPSRFSGSGSGTEFTLTISSLQPEDFATYICQQYNYYPYTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGECSEQ ID NO: 54-Humanized light chain nucleic acid sequenceatgggctggagctggatcctgctgttcctcctgagcgtgacagcaggagtgcacagcgacatccagctgacccagagcccttccttcctgagcacaagcgtcggagatagagtcacaatcacctgtaaagcaagccagaacgtgggccccaacgtggcatggtacaggcagaagcccgggaaaagccctaaggccctgatctattctgctagttaccggtattctggcgtcccaagcagattctcaggcagcgggtccggaactgagtttaccctgacaatcagctccctgcagcccgaagacttcgccacctacatttgtcagcagtacaactattacccatacaccttcgggcaggggaccaaactggaaatcaagcgaacggtggctgcaccatctgtcttcatcttcccgccatctgatgagcagttgaaatctggaactgcctctgttgtgtgcctgctgaataacttctatcccagagaggccaaagtacagtggaaggtggataacgccctccaatcgggtaactcccaggagagtgtcacagagcaggacagcaaggacagcacctacagcctcagcagcaccctgacgctgagcaaagcagactacgagaaacacaaagtctacgcctgcgaagtcacccatcagggcctgagctcgcccgtcacaaagagcttcaacaggggagagtgt SEQ ID NO: 55-Humanized light chain amino acid sequenceMGWSWILLFLLSVTAGVHSDIQMTQSPSFLSTSVGDRVTVTCKASQNVGPNVAWYRQKPGKSPKALIYSASYRYSGVPSRFSGSGSGTEFTLTISSVQPEDFATYICQQYNYYPYTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGECSEQ ID NO: 56-Humanized light chain nucleic acid sequenceatgggctggagctggatcctgctgttcctcctgagcgtgacagcaggagtgcacagcgacatccagatgacccagtccccctccttcctgagcacaagcgtgggcgatagagtcaccgtcacctgtaaagcaagccagaacgtgggccccaacgtggcatggtacaggcagaagcccgggaaaagccctaaggccctgatctattctgctagttaccggtattctggcgtgccaagcagattctcaggcagcgggtccggaactgagtttaccctgacaatcagctccgtccagcccgaagacttcgccacctacatttgtcagcagtacaactattacccatacaccttcgggcaggggactaaactggaaatcaagcgaacggtggctgcaccatctgtcttcatcttcccgccatctgatgagcagttgaaatctggaactgcctctgttgtgtgcctgctgaataacttctatcccagagaggccaaagtacagtggaaggtggataacgccctccaatcgggtaactcccaggagagtgtcacagagcaggacagcaaggacagcacctacagcctcagcagcaccctgacgctgagcaaagcagactacgagaaacacaaagtctacgcctgcgaagtcacccatcagggcctgagctcgcccgtcacaaagagcttcaacaggggagagtgt SEQ ID NO: 57-Light chain constant region amino acid sequenceRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGECSEQ ID NO: 58-Light chain constant region amino acid sequenceGQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADSSPVKAGVETTTPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTECSSEQ ID NO: 59-Heavy chain constant region amino acid sequenceASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGKSEQ ID NO: 60-Humanized heavy chain variable region amino acid sequenceQVQLVQSGAEVKKPGASVKVSCKASGYTFTDYYMNWVRQAPGQGLEWMGDVNPNNGGTHYNQKFKGRVTMTRDTSISTAYMELSRLRSDDTAVYYCARITIVPVYFDFWGQGTLVTVSSSEQ ID NO: 61-Humanized light chain variable region amino acid sequenceDIQLTQSPSFLSASVGDRVTITCKASQNVGPNVAWYQQKPGKAPKLLIYSASYRYSGVPSRFSGSGSGTEFTLTISSLQPEDFATYYCQQYNYYPYTFGQGTKLEIK

1-26. (canceled)
 27. An isolated antibody or antigen-binding fragmentthereof, which specifically binds calcitonin gene-related peptide (CGRP)and comprises: (a) a light chain CDR1 sequence identical, substantiallyidentical or substantially similar to a CDR1 sequence selected from SEQID NOs: 9-11; (b) a light chain CDR2 sequence identical, substantiallyidentical or substantially similar to a CDR2 sequence selected from SEQID NOs: 12-13; (c) a light chain CDR3 sequence identical, substantiallyidentical or substantially similar to a CDR3 sequence selected from SEQID NOs: 14-16; (d) a heavy chain CDR1 sequence identical, substantiallyidentical or substantially similar to a CDR1 sequence selected from SEQID NO: 2; (e) a heavy chain CDR2 sequence identical, substantiallyidentical or substantially similar to a CDR2 sequence selected from SEQID NOs: 3-5; (f) a heavy chain CDR3 sequence identical, substantiallyidentical or substantially similar to a CDR3 sequence selected from SEQID NOs: 6-8.
 28. An isolated antibody or antigen-binding fragmentthereof according to claim 27, which comprises: (a) a light chain CDR1sequence identical, substantially identical or substantially similar toSEQ ID NO: 9; (b) a light chain CDR2 sequence identical, substantiallyidentical or substantially similar to SEQ ID NO: 12; (c) a light chainCDR3 sequence identical, substantially identical or substantiallysimilar to SEQ ID NO: 14; (d) a heavy chain CDR1 sequence identical,substantially identical or substantially similar to SEQ ID NO: 2; (e) aheavy chain CDR2 sequence identical, substantially identical orsubstantially similar to SEQ ID NO: 3; and (f) a heavy chain CDR3sequence identical, substantially identical or substantially similar toSEQ ID NO:
 6. 29. An isolated antibody or antigen-binding fragmentthereof according to claim 27, which comprises: (a) a light chain CDR1sequence identical, substantially identical or substantially similar toSEQ ID NO: 10; (b) a light chain CDR2 sequence identical, substantiallyidentical or substantially similar to SEQ ID NO: 13; (c) a light chainCDR3 sequence identical, substantially identical or substantiallysimilar to SEQ ID NO: 15; (d) a heavy chain CDR1 sequence identical,substantially identical or substantially similar to SEQ ID NO: 2; (e) aheavy chain CDR2 sequence identical, substantially identical orsubstantially similar to SEQ ID NO: 4; and (f) a heavy chain CDR3sequence identical, substantially identical or substantially similar toSEQ ID NO:
 7. 30. An isolated antibody or antigen-binding fragmentthereof according to claim 27, which comprises: (a) a light chain CDR1sequence identical, substantially identical or substantially similar toSEQ ID NO: 11; (b) a light chain CDR2 sequence identical, substantiallyidentical or substantially similar to SEQ ID NO: 13; (c) a light chainCDR3 sequence identical, substantially identical or substantiallysimilar to SEQ ID NO: 16; (d) a heavy chain CDR1 sequence identical,substantially identical or substantially similar to SEQ ID NO: 2; (e) aheavy chain CDR2 sequence identical, substantially identical orsubstantially similar to SEQ ID NO: 5; and (f) a heavy chain CDR3sequence identical, substantially identical or substantially similar toSEQ ID NO:
 8. 31. An isolated antibody or antigen-binding fragmentthereof according to claim 27, further comprising a set of four variableregion framework regions from a human immunoglobulin (IgG).
 32. Anisolated antibody or antigen-binding fragment thereof according to claim27 that binds to CGRP protein with a dissociation constant (K_(D)) of atleast about 1×10⁻⁶ M, at least about 1×10⁻⁷ M, at least about 1×10⁻⁸ M,at least about 1×10⁻⁹ M, at least about 1×10⁻¹⁰ M, at least about1×10⁻¹¹ M, or at least about 1×10⁻¹² M.
 33. An isolated antibody orantigen-binding fragment thereof according to claim 27 wherein theantibody or antigen-binding fragment is selected from a human antibody,a humanized antibody, chimeric antibody, a monoclonal antibody, apolyclonal antibody, a recombinant antibody, an antigen-binding antibodyfragment, a single chain antibody, a diabody, a triabody, a tetrabody, aFab fragment, a Fab′ fragment, a Fab₂ fragment, a F(ab)′₂ fragment, adomain antibody, an IgD antibody, an IgE antibody, an IgM antibody, anIgG1 antibody, an IgG2 antibody, an IgG3 antibody, an IgG4 antibody, oran IgG4 antibody having at least one mutation in the hinge region thatalleviates a tendency to form intra H-chain disulfide bonds.
 34. Anisolated humanized antibody or antigen-binding fragment thereofaccording to claim 33 which comprises a heavy chain variable regionsequence identical, substantially identical or substantially similar toSEQ ID NOs: 33, 35, 37 and 39, and a light chain variable regionsequence identical, substantially identical or substantially similar toSEQ ID NOs: 34, 36, 38 and
 40. 35. An isolated humanized antibody orantigen-binding fragment thereof according to claim 33 which comprises aheavy chain sequence identical, substantially identical or substantiallysimilar to SEQ ID NOs: 43, 45, 47 and 49, and a light chain sequenceidentical, substantially identical or substantially similar to SEQ IDNOs: 49, 51, 53 and
 55. 36. An isolated humanized antibody orantigen-binding fragment thereof according to claim 27 which comprisesthe heavy chain sequence set forth in SEQ ID NO: 45, and the light chainsequence set forth in SEQ ID NO:
 51. 37. An isolated humanized antibodyor antigen-binding fragment thereof according to claim 27 whichcomprises the heavy chain sequence set forth in SEQ ID NO: 45, and thelight chain sequence set forth in SEQ ID NO:
 53. 38. An isolatedhumanized antibody or antigen-binding fragment thereof according toclaim 27 which comprises the heavy chain sequence set forth in SEQ IDNO: 47, and the light chain sequence set forth in SEQ ID NO:
 53. 39. Anisolated humanized antibody or antigen-binding fragment thereofaccording to claim 27 which comprises the heavy chain sequence set forthin SEQ ID NO: 47, and the light chain sequence set forth in SEQ ID NO:51.
 40. A pharmaceutical composition comprising an isolated antibody orantigen-binding fragment thereof according to claim 27 in admixture witha pharmaceutically acceptable carrier.
 41. A method of treating asubject suffering from a CGRP-associated disorder, comprisingadministering to said subject a therapeutically effective amount of thepharmaceutical composition of claim
 39. 42. A method according to claim40, wherein the CGRP-associated disorder is selected from the groupconsisting of headaches, hot flushes, chronic pain, type II diabetesmellitus, functional bowel disorders or an inflammatory bowel diseases,diarrhea, psoriasis, pain and itch associated with arthritis and skindisease, cardiovascular disorders, and hemodynamic derangementassociated with endotoxemia, sepsis, obesity, diabetes and arthritis.43. A method of treating a subject suffering from a CGRP-associateddisorder, comprising administering to said subject a) a therapeuticallyeffective amount of a pharmaceutical composition of claim 39; and b) oneor more additional agent useful for treating the specificCGRP-associated disorder.
 44. An isolated nucleic acid comprising apolynucleotide sequence encoding an antibody or antigen-binding fragmentthereof according to claim
 27. 45. A recombinant expression vectorcomprising the isolated nucleic acid of claim
 44. 46. A host cellcomprising the vector of claim 45.